LOCUS Exported 7018 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION individual 6N barcode to make barcoded cell line variants. ACCESSION . VERSION . KEYWORDS MSCV-6NBC-GFP-PGK-Puro-Barcode47 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7018 /organism="synthetic DNA construct" /mol_type="other DNA" 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" CDS 1427..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(4111..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(5224..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 7017..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 gatcccatgc cccaccatgg 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 //