LOCUS       Complete_example-CROPseq-multi-v2-palmitoyl-mTagBFP2_(tRNA_Q)        9189 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1509.
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..2995
                     /standard_name="palmitoylation signal"
     CDS             2996..3706
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /note="mammalian codon-optimized"
                     /standard_name="mTagBFP2"
     CDS             3707..3709
                     /standard_name="STOP"
     misc_feature    3725..4313
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    4364..4383
                     /standard_name="PPT"
     misc_feature    join(4385..>4419,5158..5338)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      4448..4465
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(4469..4474)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(4475..4572)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    4542..4553
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(4573..4592)
                     /standard_name="spacer 2"
     tRNA            complement(4593..4664)
                     /standard_name="tRNA_Q"
     tRNA            complement(4665..4670)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(4671..4770)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    4740..4751
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(4771..4790)
                     /standard_name="spacer 1"
     tRNA            complement(4791..4861)
                     /standard_name="Gly tRNA"
     misc_difference complement(4862)
                     /standard_name="5'G"
     regulatory      complement(4863..5111)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    5416..5537
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      5577..5712
     misc_feature    complement(5733..5751)
                     /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      5919..6374
     regulatory      6400..6504
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6505..7365
                     /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      7536..8124
     protein_bind    8412..8433
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      8448..8478
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    8486..8502
                     /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      8547..8565
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      8593..8972
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      8974..9189
                     /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 ccaccatgct
     2941 gtgctgtatg agaagaacca aacaggttga aaagaatgat gaggaccaaa agatcatggt
     3001 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     3061 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     3121 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     3181 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     3241 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgagga
     3301 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     3361 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     3421 cggctgggag gccttcaccg aaacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     3481 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata
     3541 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     3601 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     3661 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaaa cgcgttaagt
     3721 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3781 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3841 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3901 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3961 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     4021 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4081 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     4141 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4201 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4261 tgttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4321 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4381 ggactggaag ggctaattca ctcccaacga agagaagatt tcaccattat cgtttcagac
     4441 ccatttttaa tacgactcac tatagggaaa aaaagcaccg actcggtgcc actttttcaa
     4501 gttgataacg gactagcctt atttcaactt gctatgctgt tnnnnnnnnn nnntccagca
     4561 tagctctgaa acnnnnnnnn nnnnnnnnnn nnaggttcca ccgagatttg aactcggatc
     4621 gctggattca gagtccagag tgctaaccat tacaccatgg aacctttgtt gcagccgact
     4681 cggctgccac ttttccaagt tgtgtacgga ccagccttat ttgaacttgc taagcagtcn
     4741 nnnnnnnnnn ntcctgctta gctctcaaac nnnnnnnnnn nnnnnnnnnn tgcattggcc
     4801 gggaatcgaa cccgggcctc ccgcgtggca ggcgagaatt ctaccactga accaccaatg
     4861 ccggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt
     4921 acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga
     4981 aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta
     5041 attctaatta tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga
     5101 aataggccct caagcttggc gtaactagat cttgagacac tgctttttgc ttgtactggg
     5161 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5221 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5281 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     5341 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     5401 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5461 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5521 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     5581 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5641 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5701 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     5761 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5821 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5881 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     6001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6061 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6361 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6421 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6481 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6541 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6601 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6661 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6721 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6781 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6841 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6901 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6961 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     7021 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     7081 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     7141 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     7201 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     7261 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     7321 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7381 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7441 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7501 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7561 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7621 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7681 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7741 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7801 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7861 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7921 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7981 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     8041 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     8101 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     8161 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     8221 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     8281 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     8341 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     8401 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     8461 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     8521 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     8581 ctgcaagctt aagacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     8641 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     8701 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     8761 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     8821 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     8881 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     8941 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     9001 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     9061 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     9121 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
     9181 gcctgtact
//
LOCUS       Complete_example-CROPseq-multi-v2-palmitoyl-mTagBFP2_(tRNA_P)        9189 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1509.
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..2995
                     /standard_name="palmitoylation signal"
     CDS             2996..3706
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /note="mammalian codon-optimized"
                     /standard_name="mTagBFP2"
     CDS             3707..3709
                     /standard_name="STOP"
     misc_feature    3725..4313
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    4364..4383
                     /standard_name="PPT"
     misc_feature    join(4385..>4419,5158..5338)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      4448..4465
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(4469..4474)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(4475..4572)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    4542..4553
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(4573..4592)
                     /standard_name="spacer 2"
     tRNA            complement(4593..4664)
                     /standard_name="tRNA_P"
     tRNA            complement(4665..4670)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(4671..4770)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    4740..4751
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(4771..4790)
                     /standard_name="spacer 1"
     tRNA            complement(4791..4861)
                     /standard_name="Gly tRNA"
     misc_difference complement(4862)
                     /standard_name="5'G"
     regulatory      complement(4863..5111)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    5416..5537
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      5577..5712
     misc_feature    complement(5733..5751)
                     /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      5919..6374
     regulatory      6400..6504
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6505..7365
                     /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      7536..8124
     protein_bind    8412..8433
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      8448..8478
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    8486..8502
                     /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      8547..8565
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      8593..8972
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      8974..9189
                     /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 ccaccatgct
     2941 gtgctgtatg agaagaacca aacaggttga aaagaatgat gaggaccaaa agatcatggt
     3001 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     3061 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     3121 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     3181 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     3241 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgagga
     3301 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     3361 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     3421 cggctgggag gccttcaccg aaacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     3481 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata
     3541 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     3601 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     3661 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaaa cgcgttaagt
     3721 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3781 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3841 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3901 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3961 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     4021 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4081 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     4141 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4201 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4261 tgttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4321 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4381 ggactggaag ggctaattca ctcccaacga agagaagatt tcaccattat cgtttcagac
     4441 ccatttttaa tacgactcac tatagggaaa aaaagcaccg actcggtgcc actttttcaa
     4501 gttgataacg gactagcctt atttcaactt gctatgctgt tnnnnnnnnn nnntccagca
     4561 tagctctgaa acnnnnnnnn nnnnnnnnnn nngggctcgt ccgggatttg aacccgggac
     4621 ctctcgcacc ctaagcgaga atcatacccc tagaccaacg agcctttgtt gcagccgact
     4681 cggctgccac ttttccaagt tgtgtacgga ccagccttat ttgaacttgc taagcagtcn
     4741 nnnnnnnnnn ntcctgctta gctctcaaac nnnnnnnnnn nnnnnnnnnn tgcattggcc
     4801 gggaatcgaa cccgggcctc ccgcgtggca ggcgagaatt ctaccactga accaccaatg
     4861 ccggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt
     4921 acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga
     4981 aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta
     5041 attctaatta tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga
     5101 aataggccct caagcttggc gtaactagat cttgagacac tgctttttgc ttgtactggg
     5161 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5221 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5281 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     5341 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     5401 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5461 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5521 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     5581 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5641 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5701 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     5761 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5821 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5881 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     6001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6061 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6361 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6421 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6481 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6541 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6601 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6661 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6721 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6781 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6841 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6901 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6961 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     7021 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     7081 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     7141 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     7201 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     7261 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     7321 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7381 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7441 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7501 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7561 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7621 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7681 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7741 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7801 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7861 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7921 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7981 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     8041 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     8101 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     8161 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     8221 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     8281 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     8341 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     8401 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     8461 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     8521 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     8581 ctgcaagctt aagacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     8641 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     8701 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     8761 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     8821 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     8881 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     8941 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     9001 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     9061 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     9121 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
     9181 gcctgtact
//
LOCUS       Complete_example-CROPseq-multi-v2-palmitoyl-mTagBFP2_(tRNA_A)        9189 bp ds-DNA     circular SYN 25-FEB-2025
DEFINITION  dual guides cloned into pRW1509.
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..2995
                     /standard_name="palmitoylation signal"
     CDS             2996..3706
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /note="mammalian codon-optimized"
                     /standard_name="mTagBFP2"
     CDS             3707..3709
                     /standard_name="STOP"
     misc_feature    3725..4313
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /standard_name="WPRE"
     misc_feature    4364..4383
                     /standard_name="PPT"
     misc_feature    join(4385..>4419,5158..5338)
                     /note="Geneious type: LTR"
                     /standard_name="3' LTR (truncated)"
     regulatory      4448..4465
                     /note="Geneious type: promoter"
                     /standard_name="T7 promoter"
     regulatory      complement(4469..4474)
                     /note="Geneious type: terminator"
                     /standard_name="U6 term"
     misc_RNA        complement(4475..4572)
                     /standard_name="sgRNA scaffold 2 + iBAR 2"
     misc_feature    4542..4553
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 2"
     misc_RNA        complement(4573..4592)
                     /standard_name="spacer 2"
     tRNA            complement(4593..4664)
                     /standard_name="tRNA_A"
     tRNA            complement(4665..4670)
                     /standard_name="tRNA 5'leader"
     misc_RNA        complement(4671..4770)
                     /standard_name="sgRNA scaffold 1 + iBAR 1"
     misc_feature    4740..4751
                     /note="Geneious type: Barcode"
                     /standard_name="iBAR 1"
     misc_RNA        complement(4771..4790)
                     /standard_name="spacer 1"
     tRNA            complement(4791..4861)
                     /standard_name="Gly tRNA"
     misc_difference complement(4862)
                     /standard_name="5'G"
     regulatory      complement(4863..5111)
                     /note="Geneious type: promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
                     /standard_name="U6 promoter"
     misc_feature    5416..5537
                     /note="Geneious type: polyA_signal"
                     /note="SV40 polyadenylation signal"
                     /standard_name="SV40 poly(A) signal"
     rep_origin      5577..5712
     misc_feature    complement(5733..5751)
                     /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      5919..6374
     regulatory      6400..6504
                     /note="Geneious type: promoter"
                     /gene="bla"
                     /standard_name="AmpR promoter"
     CDS             6505..7365
                     /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      7536..8124
     protein_bind    8412..8433
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /standard_name="CAP binding site"
     regulatory      8448..8478
                     /note="Geneious type: promoter"
                     /note="promoter for the E. coli lac operon"
                     /standard_name="lac promoter"
     protein_bind    8486..8502
                     /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      8547..8565
                     /note="Geneious type: promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /standard_name="T3 promoter"
     regulatory      8593..8972
                     /note="Geneious type: enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
                     /standard_name="CMV enhancer"
     regulatory      8974..9189
                     /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 ccaccatgct
     2941 gtgctgtatg agaagaacca aacaggttga aaagaatgat gaggaccaaa agatcatggt
     3001 gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     3061 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     3121 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct
     3181 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga
     3241 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgagga
     3301 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     3361 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     3421 cggctgggag gccttcaccg aaacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     3481 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca agaccacata
     3541 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     3601 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     3661 ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaaa cgcgttaagt
     3721 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3781 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3841 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     3901 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     3961 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     4021 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     4081 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     4141 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     4201 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     4261 tgttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
     4321 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4381 ggactggaag ggctaattca ctcccaacga agagaagatt tcaccattat cgtttcagac
     4441 ccatttttaa tacgactcac tatagggaaa aaaagcaccg actcggtgcc actttttcaa
     4501 gttgataacg gactagcctt atttcaactt gctatgctgt tnnnnnnnnn nnntccagca
     4561 tagctctgaa acnnnnnnnn nnnnnnnnnn nntggaggta ctggggatcg aacccaggac
     4621 ctcgtgcatg ctaagcacgc gctctaccac tgagctatac cccctttgtt gcagccgact
     4681 cggctgccac ttttccaagt tgtgtacgga ccagccttat ttgaacttgc taagcagtcn
     4741 nnnnnnnnnn ntcctgctta gctctcaaac nnnnnnnnnn nnnnnnnnnn tgcattggcc
     4801 gggaatcgaa cccgggcctc ccgcgtggca ggcgagaatt ctaccactga accaccaatg
     4861 ccggtgtttc gtcctttcca caagatatat aaagccaaga aatcgaaata ctttcaagtt
     4921 acggtaagca tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga
     4981 aattattact ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta
     5041 attctaatta tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga
     5101 aataggccct caagcttggc gtaactagat cttgagacac tgctttttgc ttgtactggg
     5161 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5221 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5281 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     5341 acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     5401 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5461 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5521 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     5581 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5641 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     5701 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     5761 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5821 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5881 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5941 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     6001 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     6061 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     6121 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     6181 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     6241 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     6301 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     6361 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6421 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6481 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6541 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6601 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6661 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6721 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6781 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6841 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6901 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6961 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     7021 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     7081 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     7141 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     7201 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     7261 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     7321 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7381 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7441 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7501 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7561 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7621 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7681 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7741 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7801 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7861 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7921 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7981 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     8041 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     8101 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     8161 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     8221 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     8281 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     8341 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     8401 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     8461 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     8521 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     8581 ctgcaagctt aagacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     8641 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     8701 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     8761 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     8821 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     8881 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     8941 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     9001 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     9061 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     9121 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
     9181 gcctgtact
//
