LOCUS       Complete_example-CROPseq-multi-v2-Puro_(tRNA_Q)        9015 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1484.
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="packaging signal of human immunodeficiency virus 
                     type 1"
                     /standard_name="HIV-1 Psi"
     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..3535
                     /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"
     misc_feature    3551..4139
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    4190..4209
                     /standard_name="PPT"
     misc_feature    join(4211..>4245,4984..5164)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      4274..4291
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(4295..4300)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(4301..4398)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    4368..4379
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(4399..4418)
                     /standard_name="spacer 2"
     tRNA            complement(4419..4490)
                     /standard_name="tRNA_Q"
     tRNA            complement(4491..4496)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(4497..4596)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    4566..4577
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(4597..4616)
                     /standard_name="spacer 1"
     tRNA            complement(4617..4687)
                     /standard_name="Gly tRNA"
     misc_difference complement(4688)
                     /standard_name="5'G"
     regulatory      complement(4689..4937)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    5242..5363
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      5403..5538
     misc_feature    complement(5559..5577)
                     /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      5745..6200
     regulatory      6226..6330
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6331..7191
                     /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      7362..7950
     protein_bind    8238..8259
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      8274..8304
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    8312..8328
                     /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      8373..8391
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      8419..8798
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      8800..9015
                     /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 ccaccatgac
     2941 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     3001 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3061 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3121 caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt
     3181 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3241 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3301 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3361 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3421 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3481 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     3541 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3601 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3661 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3721 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3781 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3841 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3901 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3961 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     4021 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     4081 tccgcgtgtt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     4141 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4201 aaggggggac tggaagggct aattcactcc caacgaagag aagatttcac cattatcgtt
     4261 tcagacccat ttttaatacg actcactata gggaaaaaaa gcaccgactc ggtgccactt
     4321 tttcaagttg ataacggact agccttattt caacttgcta tgctgttnnn nnnnnnnnnt
     4381 ccagcatagc tctgaaacnn nnnnnnnnnn nnnnnnnnag gttccaccga gatttgaact
     4441 cggatcgctg gattcagagt ccagagtgct aaccattaca ccatggaacc tttgttgcag
     4501 ccgactcggc tgccactttt ccaagttgtg tacggaccag ccttatttga acttgctaag
     4561 cagtcnnnnn nnnnnnntcc tgcttagctc tcaaacnnnn nnnnnnnnnn nnnnnntgca
     4621 ttggccggga atcgaacccg ggcctcccgc gtggcaggcg agaattctac cactgaacca
     4681 ccaatgccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt
     4741 caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac
     4801 ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc
     4861 aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc
     4921 atgggaaata ggccctcaag cttggcgtaa ctagatcttg agacactgct ttttgcttgt
     4981 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     5041 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     5101 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     5161 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     5221 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     5281 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5341 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     5401 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5461 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5521 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     5581 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5641 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5701 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     5761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6061 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6121 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6181 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8041 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     8101 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     8161 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     8221 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     8281 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     8341 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     8401 ctggagctgc aagcttaaga cattgattat tgactagtta ttaatagtaa tcaattacgg
     8461 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     8521 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     8581 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     8641 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     8701 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     8761 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     8821 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     8881 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     8941 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg
     9001 cgttttgcct gtact
//
LOCUS       Complete_example-CROPseq-multi-v2-Puro_(tRNA_P)        9015 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1484.
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="packaging signal of human immunodeficiency virus 
                     type 1"
                     /standard_name="HIV-1 Psi"
     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..3535
                     /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"
     misc_feature    3551..4139
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    4190..4209
                     /standard_name="PPT"
     misc_feature    join(4211..>4245,4984..5164)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      4274..4291
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(4295..4300)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(4301..4398)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    4368..4379
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(4399..4418)
                     /standard_name="spacer 2"
     tRNA            complement(4419..4490)
                     /standard_name="tRNA_P"
     tRNA            complement(4491..4496)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(4497..4596)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    4566..4577
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(4597..4616)
                     /standard_name="spacer 1"
     tRNA            complement(4617..4687)
                     /standard_name="Gly tRNA"
     misc_difference complement(4688)
                     /standard_name="5'G"
     regulatory      complement(4689..4937)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    5242..5363
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      5403..5538
     misc_feature    complement(5559..5577)
                     /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      5745..6200
     regulatory      6226..6330
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6331..7191
                     /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      7362..7950
     protein_bind    8238..8259
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      8274..8304
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    8312..8328
                     /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      8373..8391
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      8419..8798
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      8800..9015
                     /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 ccaccatgac
     2941 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     3001 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3061 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3121 caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt
     3181 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3241 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3301 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3361 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3421 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3481 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     3541 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3601 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3661 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3721 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3781 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3841 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3901 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3961 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     4021 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     4081 tccgcgtgtt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     4141 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4201 aaggggggac tggaagggct aattcactcc caacgaagag aagatttcac cattatcgtt
     4261 tcagacccat ttttaatacg actcactata gggaaaaaaa gcaccgactc ggtgccactt
     4321 tttcaagttg ataacggact agccttattt caacttgcta tgctgttnnn nnnnnnnnnt
     4381 ccagcatagc tctgaaacnn nnnnnnnnnn nnnnnnnngg gctcgtccgg gatttgaacc
     4441 cgggacctct cgcaccctaa gcgagaatca tacccctaga ccaacgagcc tttgttgcag
     4501 ccgactcggc tgccactttt ccaagttgtg tacggaccag ccttatttga acttgctaag
     4561 cagtcnnnnn nnnnnnntcc tgcttagctc tcaaacnnnn nnnnnnnnnn nnnnnntgca
     4621 ttggccggga atcgaacccg ggcctcccgc gtggcaggcg agaattctac cactgaacca
     4681 ccaatgccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt
     4741 caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac
     4801 ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc
     4861 aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc
     4921 atgggaaata ggccctcaag cttggcgtaa ctagatcttg agacactgct ttttgcttgt
     4981 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     5041 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     5101 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     5161 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     5221 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     5281 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5341 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     5401 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5461 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5521 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     5581 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5641 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5701 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     5761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6061 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6121 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6181 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8041 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     8101 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     8161 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     8221 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     8281 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     8341 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     8401 ctggagctgc aagcttaaga cattgattat tgactagtta ttaatagtaa tcaattacgg
     8461 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     8521 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     8581 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     8641 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     8701 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     8761 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     8821 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     8881 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     8941 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg
     9001 cgttttgcct gtact
//
LOCUS       Complete_example-CROPseq-multi-v2-Puro_(tRNA_A)        9015 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1484.
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="packaging signal of human immunodeficiency virus 
                     type 1"
                     /standard_name="HIV-1 Psi"
     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..3535
                     /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"
     misc_feature    3551..4139
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    4190..4209
                     /standard_name="PPT"
     misc_feature    join(4211..>4245,4984..5164)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      4274..4291
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(4295..4300)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(4301..4398)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    4368..4379
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(4399..4418)
                     /standard_name="spacer 2"
     tRNA            complement(4419..4490)
                     /standard_name="tRNA_A"
     tRNA            complement(4491..4496)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(4497..4596)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    4566..4577
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(4597..4616)
                     /standard_name="spacer 1"
     tRNA            complement(4617..4687)
                     /standard_name="Gly tRNA"
     misc_difference complement(4688)
                     /standard_name="5'G"
     regulatory      complement(4689..4937)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    5242..5363
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      5403..5538
     misc_feature    complement(5559..5577)
                     /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      5745..6200
     regulatory      6226..6330
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6331..7191
                     /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      7362..7950
     protein_bind    8238..8259
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      8274..8304
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    8312..8328
                     /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      8373..8391
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      8419..8798
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      8800..9015
                     /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 ccaccatgac
     2941 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     3001 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3061 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3121 caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt
     3181 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3241 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3301 gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3361 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3421 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3481 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg
     3541 ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     3601 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     3661 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     3721 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     3781 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     3841 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     3901 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     3961 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     4021 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     4081 tccgcgtgtt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg
     4141 tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     4201 aaggggggac tggaagggct aattcactcc caacgaagag aagatttcac cattatcgtt
     4261 tcagacccat ttttaatacg actcactata gggaaaaaaa gcaccgactc ggtgccactt
     4321 tttcaagttg ataacggact agccttattt caacttgcta tgctgttnnn nnnnnnnnnt
     4381 ccagcatagc tctgaaacnn nnnnnnnnnn nnnnnnnntg gaggtactgg ggatcgaacc
     4441 caggacctcg tgcatgctaa gcacgcgctc taccactgag ctataccccc tttgttgcag
     4501 ccgactcggc tgccactttt ccaagttgtg tacggaccag ccttatttga acttgctaag
     4561 cagtcnnnnn nnnnnnntcc tgcttagctc tcaaacnnnn nnnnnnnnnn nnnnnntgca
     4621 ttggccggga atcgaacccg ggcctcccgc gtggcaggcg agaattctac cactgaacca
     4681 ccaatgccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt
     4741 caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac
     4801 ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc
     4861 aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc
     4921 atgggaaata ggccctcaag cttggcgtaa ctagatcttg agacactgct ttttgcttgt
     4981 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     5041 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     5101 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     5161 agcagtacgt atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     5221 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     5281 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5341 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     5401 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     5461 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     5521 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     5581 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5641 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5701 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     5761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6061 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6121 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6181 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8041 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     8101 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     8161 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     8221 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     8281 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     8341 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     8401 ctggagctgc aagcttaaga cattgattat tgactagtta ttaatagtaa tcaattacgg
     8461 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     8521 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     8581 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     8641 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     8701 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     8761 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     8821 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     8881 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     8941 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg
     9001 cgttttgcct gtact
//
