LOCUS Exported 4795 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Donor empty vector with floxed puromycin resistance gene expression cassette. ACCESSION . VERSION . KEYWORDS PL552 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4795) AUTHORS Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, Huang CT, Du Z, Zhang SC TITLE Engineering Human Stem Cell Lines with Inducible Gene Knockout using CRISPR/Cas9. JOURNAL Cell Stem Cell. 2015 Jul 1. pii: S1934-5909(15)00261-1. doi: 10.1016/j.stem.2015.06.001. PUBMED 26145478 REFERENCE 2 (bases 1 to 4795) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4795 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..19 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" protein_bind complement(88..121) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 182..681 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(217..240) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 614..633 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 721..1320 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(721..740) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" protein_bind complement(1877..1910) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1936..1952) /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1936..1952) /label=pBluescriptSK /note="For pBluescript vector" primer_bind complement(1984..2003) /label=T7 /note="T7 promoter, forward primer" promoter complement(1985..2003) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2010..2027) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(2010..2026) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2019..2041) /label=M13/pUC Forward /note="In lacZ gene" rep_origin complement(2167..2622) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2304..2325) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2516..2535 /label=F1ori-R /note="F1 origin, reverse primer" promoter 2649..2753 /gene="bla" /label=AmpR promoter CDS 2754..3614 /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 complement(2972..2991) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3785..4373 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4274..4293 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4527..4544 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4661..4682 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4697..4727 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4735..4751 /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)." primer_bind 4740..4762 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4759..4775 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4759..4775 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" ORIGIN 1 aattaaccct cactaaaggg aacaaaagct ggtaccgggc cccccctcga ggtcgacgaa 61 ttccgatcat attcaataac ccttaatata acttcgtata atgtatgcta tacgaagtta 121 ttaggtctga agaggagttt acgtccagcc aagcttagga tctcgacctc gaaattctac 181 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg 241 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg 301 cgccaaccga ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc 361 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca 421 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt 481 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg 541 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc 601 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct 661 tcctcatctc cgggcctttc gacctgcatc catctagatc tcgagcagct gaagcttacc 721 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta 781 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac 841 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac 901 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag 961 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt 1021 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag 1081 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc 1141 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg 1201 gagacctccg cgccccacaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc 1261 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga 1321 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcgatg 1381 atatcagatc cccgggatgc agaaattgat gatctattaa acaataaaga tgtccactaa 1441 aatggaagtt tttcctgtca tactttgtta agaagggtga gaacagagta cctacatttt 1501 gaatggaagg attggagcta cgggggtggg ggtggggtgg gattagataa atgcctgctc 1561 tttactgaag gctctttact attgctttat gataatgttt catagttgga tatcataatt 1621 taaacaagca aaaccaaatt aagggccagc tcattcctcc cactcatgat ctatagatct 1681 atagatctct cgtgggatca ttgtttttct cttgattccc actttgtggt tctaagtact 1741 gtggtttcca aatgtgtcag tttcatagcc tgaagaacga gatcagcagc ctctgttcca 1801 catacacttc attctcagta ttgttttgcc aagttctaat tccatcagaa gctggtcgag 1861 atccggaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtccctc 1921 gaagaggttc actaggatcc actagttcta gagcggccgc caccgcggtg gagctccaat 1981 tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg 2041 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 2101 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 2161 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 2221 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 2281 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 2341 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 2401 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 2461 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 2521 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 2581 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg 2641 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 2701 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 2761 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 2821 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 2881 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 2941 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 3001 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3061 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3121 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3181 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 3241 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 3301 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 3361 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 3421 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 3481 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 3541 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 3601 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 3661 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 3721 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 3781 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 3841 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 3901 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 3961 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4021 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4081 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4141 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 4201 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4261 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 4321 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 4381 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 4441 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 4501 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 4561 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 4621 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 4681 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 4741 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcga //