LOCUS       sequence-159850-        7018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  individual 6N barcode to make barcoded cell line variants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7018)
  AUTHORS   Gruner BM, Schulze CJ, Yang D, Ogasawara D, Dix MM, Rogers ZN, 
            Chuang CH, McFarland CD, Chiou SH, Brown JM, Cravatt BF, Bogyo M, 
            Winslow MM
  TITLE     An in vivo multiplexed small-molecule screening platform.
  JOURNAL   Nat Methods. 2016 Sep 12. doi: 10.1038/nmeth.3992.
  PUBMED    27617390
REFERENCE   2  (bases 1 to 7018)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2016 Sep 12. doi: 10.1038/nmeth.3992."
COMMENT     Sequence Label: MSCV-6NBC-GFP-PGK-Puro-Barcode32
FEATURES             Location/Qualifiers
     source          1..7018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1421..1430
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1427..1429,1430..1432,1433..2146)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1427..1429,1430..1432,1433..2143)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1472..1493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1733..1752)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2080..2101
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        2161..2660
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2196..2219)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2593..2612
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2681..3280
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2681..2700)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3177..3197
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3380..3894
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(4063..4079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4063..4079)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4076..4098)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4087..4103
                     /label=lac operator
                     /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)."
     promoter        complement(join(4111..4117,4118..4135,4136..4141))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4156..4177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4294..4311)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4465..5053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4545..4564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5224..6015,6016..6084))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5847..5866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6085..6189)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6257..6275
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6313..6335)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6435..6454
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6648..6670
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6798..6817
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7017..7018,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatccgagac gccaccatgg tgagcaaggg
     1441 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1501 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1561 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1621 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1681 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1741 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1801 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1861 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1921 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1981 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2041 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2101 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtagcgga gaattctacc
     2161 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     2221 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     2281 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     2341 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     2401 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     2461 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     2521 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     2581 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     2641 cctcatctcc gggcctttcg acctgcagcc caagcttacc atgaccgagt acaagcccac
     2701 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     2761 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     2821 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     2881 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     2941 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3001 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3061 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3121 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3181 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3241 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     3301 gcgcccgacc gaaaggagcg cacgacccca tgcatcgata aaataaaaga ttttatttag
     3361 tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt
     3421 aacgccattt tgcaaggcat ggaaaataca taactgagaa tagagaagtt cagatcaagg
     3481 ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct
     3541 gccccggctc agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc
     3601 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
     3661 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
     3721 aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc
     3781 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
     3841 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa
     3901 cagtttcttg aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct
     3961 gactcaatta gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat
     4021 ggacagcgca gaagagctgg ggagaattaa ttcgtaatca tggtcatagc tgtttcctgt
     4081 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4141 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4201 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4261 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4321 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4381 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4441 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4501 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4561 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4621 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4681 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4741 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4801 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4861 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4921 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4981 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5041 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5101 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5161 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5221 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5281 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5341 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5401 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5461 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5521 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5581 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5641 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5701 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5761 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5821 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5881 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5941 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6001 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6061 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6121 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6181 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     6241 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     6301 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     6361 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     6421 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     6481 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct
     6541 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     6601 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     6661 ttgtaaaacg acggcgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     6721 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     6781 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     6841 ccggtgatgc cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac
     6901 aggatatcag tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata
     6961 gagtacgagc catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaa
//
