LOCUS       Complete_example-CROPseq-multi-v2-SpCas9-P2A-Puro_(tRNA_Q)       13257 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1512.
ACCESSION
VERSION     .
KEYWORDS    CROPseq-puro-v2.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 10224)
  AUTHORS   Feldman F, Singh A, Schmid-Burgk JL, Mezger A, Garrity AJ, Carlson
            RJ, Zhang F and Blainey PC
  TITLE     Pooled optical screens in human cells
  JOURNAL   bioRxiv 383943
REFERENCE   2  (bases 1 to 10224)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 4, 2019 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     misc_feature    1..181
                     /note="Geneious type: LTR"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /standard_name="5' LTR (truncated)"
     misc_feature    228..353
                     /note="pLannotate"
                     /standard_name="HIV-1 Psi"
     CDS             337..700
                     /note="pLannotate"
                     /standard_name="HIV-1 gag"
     misc_feature    846..1079
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to
                     thecytoplasm."
                     /standard_name="RRE"
     misc_feature    1489..1606
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /standard_name="cPPT/CTS"
     regulatory      1745..2923
                     /note="Geneious type: promoter"
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
                     /standard_name="EF-1-alpha promoter"
     intron          1976..2914
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
                     /standard_name="EF-1-alpha intron A"
     CDS             2936..7039
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /standard_name="SpCas9"
     CDS             7040..7087
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /standard_name="nucleoplasmin NLS"
     CDS             7088..7111
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                      cleavage site"
                     /translation="DYKDDDDK"
                     /standard_name="FLAG"
     CDS             7112..7177
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /standard_name="P2A"
     CDS             7178..7774
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR
                     LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
                     /standard_name="PuroR"
     CDS             7775..7777
                     /standard_name="STOP"
     misc_feature    7793..8381
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    8432..8451
                     /standard_name="PPT"
     misc_feature    join(8453..>8487,9226..9406)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      8516..8533
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(8537..8542)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(8543..8640)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    8610..8621
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(8641..8660)
                     /standard_name="spacer 2"
     tRNA            complement(8661..8732)
                     /standard_name="tRNA_Q"
     tRNA            complement(8733..8738)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(8739..8838)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    8808..8819
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(8839..8858)
                     /standard_name="spacer 1"
     tRNA            complement(8859..8929)
                     /standard_name="Gly tRNA"
     misc_difference complement(8930)
                     /standard_name="5'G"
     regulatory      complement(8931..9179)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    9484..9605
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      9645..9780
     misc_feature    complement(9801..9819)
                     /note="Geneious type: Marker"
                     /note="This T7 promoter is a vestigial sequence in the
                     plasmid backbone. It is outside the lentiviral LTRs, so it
                     is not encoded by the resulting lentiviral components."
                     /standard_name="(this is not the) T7 promoter (you're
                     looking for)"
     rep_origin      9987..10442
     regulatory      10468..10572
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             10573..11433
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /standard_name="AmpR"
     rep_origin      11604..12192
     protein_bind    12480..12501
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      12516..12546
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    12554..12570
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /standard_name="lac operator"
     regulatory      12615..12633
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      12661..13040
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      13042..13257
                     /note="Geneious type: promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /standard_name="CMV promoter"
ORIGIN      
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     1501 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     1561 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     1621 ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa
     1681 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     1741 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     1801 ggggggaggg gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga
     1861 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     1921 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
     1981 tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg
     2041 aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
     2101 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
     2161 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
     2221 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
     2281 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
     2341 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
     2401 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
     2461 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
     2521 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
     2581 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     2641 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     2701 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     2761 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     2821 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     2881 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg ccaccatgga
     2941 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     3001 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     3061 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     3121 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     3181 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     3241 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     3301 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     3361 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     3421 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     3481 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     3541 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     3601 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     3661 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     3721 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     3781 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     3841 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     3901 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     3961 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     4021 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     4081 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     4141 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     4201 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     4261 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     4321 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     4381 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     4441 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     4501 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     4561 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     4621 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     4681 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     4741 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     4801 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     4861 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     4921 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     4981 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     5041 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     5101 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     5161 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     5221 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     5281 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     5341 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     5401 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     5461 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     5521 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     5581 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     5641 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     5701 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     5761 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     5821 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     5881 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     5941 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     6001 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     6061 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     6121 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     6181 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     6241 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     6301 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     6361 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     6421 gctactgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     6481 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     6541 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     6601 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     6661 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     6721 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     6781 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     6841 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     6901 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     6961 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     7021 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
     7081 gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct
     7141 gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt
     7201 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     7261 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     7321 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     7381 cgacggcgcc gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     7441 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     7501 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     7561 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     7621 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     7681 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     7741 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc
     7801 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7861 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7921 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7981 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     8041 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     8101 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     8161 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     8221 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     8281 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtg ttcgccttcg
     8341 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
     8401 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     8461 ctaattcact cccaacgaag agaagatttc accattatcg tttcagaccc atttttaata
     8521 cgactcacta tagggaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     8581 ctagccttat ttcaacttgc tatgctgttn nnnnnnnnnn ntccagcata gctctgaaac
     8641 nnnnnnnnnn nnnnnnnnnn aggttccacc gagatttgaa ctcggatcgc tggattcaga
     8701 gtccagagtg ctaaccatta caccatggaa cctttgttgc agccgactcg gctgccactt
     8761 ttccaagttg tgtacggacc agccttattt gaacttgcta agcagtcnnn nnnnnnnnnt
     8821 cctgcttagc tctcaaacnn nnnnnnnnnn nnnnnnnntg cattggccgg gaatcgaacc
     8881 cgggcctccc gcgtggcagg cgagaattct accactgaac caccaatgcc ggtgtttcgt
     8941 cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata
     9001 tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt
     9061 ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat tctaattatc
     9121 tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctca
     9181 agcttggcgt aactagatct tgagacactg ctttttgctt gtactgggtc tctctggtta
     9241 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     9301 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     9361 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt
     9421 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     9481 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     9541 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     9601 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     9661 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     9721 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     9781 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     9841 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     9901 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     9961 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
    10021 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
    10081 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
    10141 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
    10201 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
    10261 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
    10321 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
    10381 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
    10441 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
    10501 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
    10561 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
    10621 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
    10681 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
    10741 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
    10801 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
    10861 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    10921 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    10981 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    11041 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    11101 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    11161 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    11221 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    11281 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    11341 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    11401 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    11461 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    11521 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    11581 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    11641 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    11701 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
    11761 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    11821 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    11881 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    11941 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    12001 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    12061 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    12121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    12181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    12241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
    12301 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
    12361 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
    12421 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
    12481 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
    12541 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
    12601 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
    12661 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
    12721 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
    12781 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
    12841 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
    12901 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
    12961 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
    13021 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
    13081 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
    13141 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
    13201 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtact
//
LOCUS       Complete_example-CROPseq-multi-v2-SpCas9-P2A-Puro_(tRNA_P)       13257 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1512.
ACCESSION
VERSION     .
KEYWORDS    CROPseq-puro-v2.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 10224)
  AUTHORS   Feldman F, Singh A, Schmid-Burgk JL, Mezger A, Garrity AJ, Carlson
            RJ, Zhang F and Blainey PC
  TITLE     Pooled optical screens in human cells
  JOURNAL   bioRxiv 383943
REFERENCE   2  (bases 1 to 10224)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 4, 2019 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     misc_feature    1..181
                     /note="Geneious type: LTR"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /standard_name="5' LTR (truncated)"
     misc_feature    228..353
                     /note="pLannotate"
                     /standard_name="HIV-1 Psi"
     CDS             337..700
                     /note="pLannotate"
                     /standard_name="HIV-1 gag"
     misc_feature    846..1079
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to
                     thecytoplasm."
                     /standard_name="RRE"
     misc_feature    1489..1606
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /standard_name="cPPT/CTS"
     regulatory      1745..2923
                     /note="Geneious type: promoter"
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
                     /standard_name="EF-1-alpha promoter"
     intron          1976..2914
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
                     /standard_name="EF-1-alpha intron A"
     CDS             2936..7039
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /standard_name="SpCas9"
     CDS             7040..7087
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /standard_name="nucleoplasmin NLS"
     CDS             7088..7111
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                      cleavage site"
                     /translation="DYKDDDDK"
                     /standard_name="FLAG"
     CDS             7112..7177
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /standard_name="P2A"
     CDS             7178..7774
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR
                     LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
                     /standard_name="PuroR"
     CDS             7775..7777
                     /standard_name="STOP"
     misc_feature    7793..8381
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    8432..8451
                     /standard_name="PPT"
     misc_feature    join(8453..>8487,9226..9406)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      8516..8533
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(8537..8542)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(8543..8640)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    8610..8621
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(8641..8660)
                     /standard_name="spacer 2"
     tRNA            complement(8661..8732)
                     /standard_name="tRNA_P"
     tRNA            complement(8733..8738)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(8739..8838)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    8808..8819
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(8839..8858)
                     /standard_name="spacer 1"
     tRNA            complement(8859..8929)
                     /standard_name="Gly tRNA"
     misc_difference complement(8930)
                     /standard_name="5'G"
     regulatory      complement(8931..9179)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    9484..9605
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      9645..9780
     misc_feature    complement(9801..9819)
                     /note="Geneious type: Marker"
                     /note="This T7 promoter is a vestigial sequence in the
                     plasmid backbone. It is outside the lentiviral LTRs, so it
                     is not encoded by the resulting lentiviral components."
                     /standard_name="(this is not the) T7 promoter (you're
                     looking for)"
     rep_origin      9987..10442
     regulatory      10468..10572
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             10573..11433
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /standard_name="AmpR"
     rep_origin      11604..12192
     protein_bind    12480..12501
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      12516..12546
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    12554..12570
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /standard_name="lac operator"
     regulatory      12615..12633
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      12661..13040
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      13042..13257
                     /note="Geneious type: promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /standard_name="CMV promoter"
ORIGIN      
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     1501 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     1561 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     1621 ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa
     1681 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     1741 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     1801 ggggggaggg gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga
     1861 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     1921 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
     1981 tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg
     2041 aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
     2101 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
     2161 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
     2221 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
     2281 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
     2341 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
     2401 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
     2461 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
     2521 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
     2581 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     2641 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     2701 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     2761 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     2821 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     2881 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg ccaccatgga
     2941 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     3001 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     3061 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     3121 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     3181 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     3241 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     3301 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     3361 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     3421 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     3481 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     3541 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     3601 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     3661 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     3721 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     3781 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     3841 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     3901 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     3961 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     4021 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     4081 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     4141 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     4201 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     4261 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     4321 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     4381 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     4441 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     4501 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     4561 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     4621 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     4681 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     4741 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     4801 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     4861 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     4921 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     4981 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     5041 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     5101 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     5161 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     5221 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     5281 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     5341 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     5401 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     5461 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     5521 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     5581 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     5641 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     5701 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     5761 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     5821 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     5881 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     5941 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     6001 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     6061 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     6121 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     6181 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     6241 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     6301 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     6361 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     6421 gctactgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     6481 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     6541 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     6601 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     6661 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     6721 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     6781 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     6841 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     6901 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     6961 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     7021 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
     7081 gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct
     7141 gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt
     7201 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     7261 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     7321 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     7381 cgacggcgcc gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     7441 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     7501 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     7561 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     7621 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     7681 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     7741 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc
     7801 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7861 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7921 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7981 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     8041 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     8101 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     8161 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     8221 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     8281 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtg ttcgccttcg
     8341 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
     8401 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     8461 ctaattcact cccaacgaag agaagatttc accattatcg tttcagaccc atttttaata
     8521 cgactcacta tagggaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     8581 ctagccttat ttcaacttgc tatgctgttn nnnnnnnnnn ntccagcata gctctgaaac
     8641 nnnnnnnnnn nnnnnnnnnn gggctcgtcc gggatttgaa cccgggacct ctcgcaccct
     8701 aagcgagaat cataccccta gaccaacgag cctttgttgc agccgactcg gctgccactt
     8761 ttccaagttg tgtacggacc agccttattt gaacttgcta agcagtcnnn nnnnnnnnnt
     8821 cctgcttagc tctcaaacnn nnnnnnnnnn nnnnnnnntg cattggccgg gaatcgaacc
     8881 cgggcctccc gcgtggcagg cgagaattct accactgaac caccaatgcc ggtgtttcgt
     8941 cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata
     9001 tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt
     9061 ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat tctaattatc
     9121 tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctca
     9181 agcttggcgt aactagatct tgagacactg ctttttgctt gtactgggtc tctctggtta
     9241 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     9301 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     9361 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt
     9421 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     9481 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     9541 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     9601 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     9661 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     9721 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     9781 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     9841 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     9901 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     9961 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
    10021 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
    10081 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
    10141 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
    10201 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
    10261 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
    10321 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
    10381 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
    10441 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
    10501 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
    10561 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
    10621 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
    10681 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
    10741 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
    10801 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
    10861 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    10921 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    10981 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    11041 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    11101 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    11161 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    11221 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    11281 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    11341 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    11401 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    11461 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    11521 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    11581 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    11641 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    11701 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
    11761 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    11821 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    11881 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    11941 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    12001 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    12061 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    12121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    12181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    12241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
    12301 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
    12361 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
    12421 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
    12481 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
    12541 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
    12601 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
    12661 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
    12721 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
    12781 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
    12841 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
    12901 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
    12961 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
    13021 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
    13081 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
    13141 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
    13201 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtact
//
LOCUS       Complete_example-CROPseq-multi-v2-SpCas9-P2A-Puro_(tRNA_A)       13257 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1512.
ACCESSION
VERSION     .
KEYWORDS    CROPseq-puro-v2.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 10224)
  AUTHORS   Feldman F, Singh A, Schmid-Burgk JL, Mezger A, Garrity AJ, Carlson
            RJ, Zhang F and Blainey PC
  TITLE     Pooled optical screens in human cells
  JOURNAL   bioRxiv 383943
REFERENCE   2  (bases 1 to 10224)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 4, 2019 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     misc_feature    1..181
                     /note="Geneious type: LTR"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /standard_name="5' LTR (truncated)"
     misc_feature    228..353
                     /note="pLannotate"
                     /standard_name="HIV-1 Psi"
     CDS             337..700
                     /note="pLannotate"
                     /standard_name="HIV-1 gag"
     misc_feature    846..1079
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to
                     thecytoplasm."
                     /standard_name="RRE"
     misc_feature    1489..1606
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /standard_name="cPPT/CTS"
     regulatory      1745..2923
                     /note="Geneious type: promoter"
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
                     /standard_name="EF-1-alpha promoter"
     intron          1976..2914
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
                     /standard_name="EF-1-alpha intron A"
     CDS             2936..7039
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK
                     KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE
                     ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH
                     MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK
                     SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD
                     NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL
                     LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK
                     LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
                     YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL
                     PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT
                     LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF
                     LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQT
                     VKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE
                     HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV
                     LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA
                     GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG
                     KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
                     MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
                     AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF
                     ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH
                     KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA
                     PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
                     /standard_name="SpCas9"
     CDS             7040..7087
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /translation="KRPAATKKAGQAKKKK"
                     /standard_name="nucleoplasmin NLS"
     CDS             7088..7111
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase
                      cleavage site"
                     /translation="DYKDDDDK"
                     /standard_name="FLAG"
     CDS             7112..7177
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding
                     separatepolypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
                     /standard_name="P2A"
     CDS             7178..7774
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR
                     LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL
                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
                     /standard_name="PuroR"
     CDS             7775..7777
                     /standard_name="STOP"
     misc_feature    7793..8381
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    8432..8451
                     /standard_name="PPT"
     misc_feature    join(8453..>8487,9226..9406)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      8516..8533
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(8537..8542)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(8543..8640)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    8610..8621
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(8641..8660)
                     /standard_name="spacer 2"
     tRNA            complement(8661..8732)
                     /standard_name="tRNA_A"
     tRNA            complement(8733..8738)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(8739..8838)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    8808..8819
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(8839..8858)
                     /standard_name="spacer 1"
     tRNA            complement(8859..8929)
                     /standard_name="Gly tRNA"
     misc_difference complement(8930)
                     /standard_name="5'G"
     regulatory      complement(8931..9179)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    9484..9605
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      9645..9780
     misc_feature    complement(9801..9819)
                     /note="Geneious type: Marker"
                     /note="This T7 promoter is a vestigial sequence in the
                     plasmid backbone. It is outside the lentiviral LTRs, so it
                     is not encoded by the resulting lentiviral components."
                     /standard_name="(this is not the) T7 promoter (you're
                     looking for)"
     rep_origin      9987..10442
     regulatory      10468..10572
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             10573..11433
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /standard_name="AmpR"
     rep_origin      11604..12192
     protein_bind    12480..12501
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      12516..12546
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    12554..12570
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /standard_name="lac operator"
     regulatory      12615..12633
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      12661..13040
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      13042..13257
                     /note="Geneious type: promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /standard_name="CMV promoter"
ORIGIN      
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa
     1501 aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat
     1561 acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag
     1621 ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggtgcaaa gatggataaa
     1681 gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga aaggagtggg
     1741 aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt
     1801 ggggggaggg gtcggcaatt gatccggtgc ctagagaagg tggcgcgggg taaactggga
     1861 aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
     1921 tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
     1981 tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg
     2041 aattacttcc acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
     2101 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
     2161 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
     2221 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
     2281 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
     2341 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
     2401 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
     2461 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
     2521 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
     2581 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     2641 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     2701 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     2761 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     2821 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     2881 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgacgtacgg ccaccatgga
     2941 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     3001 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     3061 catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac
     3121 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     3181 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     3241 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     3301 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     3361 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     3421 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     3481 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     3541 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     3601 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     3661 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     3721 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     3781 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     3841 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     3901 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     3961 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     4021 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     4081 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     4141 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     4201 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     4261 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     4321 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     4381 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     4441 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     4501 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     4561 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     4621 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     4681 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     4741 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     4801 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     4861 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     4921 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     4981 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     5041 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     5101 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     5161 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     5221 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     5281 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     5341 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     5401 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     5461 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     5521 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     5581 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     5641 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     5701 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     5761 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     5821 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca
     5881 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     5941 taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     6001 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     6061 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     6121 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     6181 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     6241 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     6301 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     6361 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     6421 gctactgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     6481 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     6541 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     6601 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     6661 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     6721 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     6781 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     6841 cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc
     6901 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     6961 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     7021 gtctcagctg ggaggcgaca agcgacctgc cgccacaaag aaggctggac aggctaagaa
     7081 gaagaaagat tacaaagacg atgacgataa gggatccggc gcaacaaact tctctctgct
     7141 gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg accgagtaca agcccacggt
     7201 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     7261 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     7321 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     7381 cgacggcgcc gccgtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     7441 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     7501 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     7561 cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     7621 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     7681 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     7741 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg acaatcaacc
     7801 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     7861 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     7921 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     7981 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     8041 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     8101 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     8161 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     8221 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     8281 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtg ttcgccttcg
     8341 ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt aagaccaatg
     8401 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     8461 ctaattcact cccaacgaag agaagatttc accattatcg tttcagaccc atttttaata
     8521 cgactcacta tagggaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     8581 ctagccttat ttcaacttgc tatgctgttn nnnnnnnnnn ntccagcata gctctgaaac
     8641 nnnnnnnnnn nnnnnnnnnn tggaggtact ggggatcgaa cccaggacct cgtgcatgct
     8701 aagcacgcgc tctaccactg agctataccc cctttgttgc agccgactcg gctgccactt
     8761 ttccaagttg tgtacggacc agccttattt gaacttgcta agcagtcnnn nnnnnnnnnt
     8821 cctgcttagc tctcaaacnn nnnnnnnnnn nnnnnnnntg cattggccgg gaatcgaacc
     8881 cgggcctccc gcgtggcagg cgagaattct accactgaac caccaatgcc ggtgtttcgt
     8941 cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac ggtaagcata
     9001 tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa ttattacttt
     9061 ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat tctaattatc
     9121 tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa taggccctca
     9181 agcttggcgt aactagatct tgagacactg ctttttgctt gtactgggtc tctctggtta
     9241 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     9301 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     9361 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtac gtatagtagt
     9421 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     9481 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     9541 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     9601 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     9661 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     9721 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     9781 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     9841 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     9901 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     9961 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
    10021 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
    10081 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
    10141 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
    10201 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
    10261 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
    10321 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
    10381 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
    10441 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
    10501 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
    10561 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
    10621 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
    10681 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
    10741 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
    10801 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
    10861 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
    10921 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
    10981 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
    11041 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
    11101 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
    11161 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
    11221 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
    11281 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
    11341 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
    11401 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    11461 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    11521 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    11581 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    11641 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    11701 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
    11761 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    11821 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    11881 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    11941 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    12001 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    12061 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    12121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    12181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    12241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
    12301 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
    12361 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
    12421 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
    12481 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
    12541 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
    12601 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
    12661 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
    12721 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
    12781 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
    12841 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
    12901 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
    12961 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
    13021 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
    13081 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
    13141 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
    13201 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtact
//
