LOCUS Exported 5801 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION donor vector backbone with constitutive eGFP. ACCESSION . VERSION . KEYWORDS pCAG-eGFP-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5801) AUTHORS Kim SI, Matsumoto T, Kagawa H, Nakamura M, Hirohata R, Ueno A, Ohishi M, Sakuma T, Soga T, Yamamoto T, Woltjen K TITLE Microhomology-assisted scarless genome editing in human iPSCs. JOURNAL Nat Commun. 2018 Mar 5;9(1):939. doi: 10.1038/s41467-018-03044-y. PUBMED 29507284 REFERENCE 2 (bases 1 to 5801) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5801 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(130..151) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 342..361 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 575..597 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 589..606 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 590..606 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 613..632 /label=T7 /note="T7 promoter, forward primer" promoter 613..631 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" enhancer 678..1057 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1060..1335 /label=chicken beta-actin promoter intron 1336..2344 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 2268..2290 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 2352..2371 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind 2438..2462 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 2468..3184 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 2468..3184 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2513..2534) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2774..2793) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3121..3142 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(3246..3270) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(3285..3301) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3286..3302) /label=pBluescriptKS /note="For pBluescript vector" primer_bind complement(3317..3334) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3323..3547 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter complement(3603..3621) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(3642..3658) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3642..3658) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3655..3677) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3666..3682 /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(3690..3720) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3735..3756 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3873..3890) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4044..4632) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4124..4143) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4803..5663) /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 5426..5445 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5664..5768) /gene="bla" /label=AmpR promoter rep_origin complement(5794..448) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" ORIGIN 1 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac 61 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg 121 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa 181 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt 241 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt 301 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga 361 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc 421 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg 481 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 541 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 601 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccggcca tgatggccga 661 attaattcaa gctagtcgac attgattatt gactagttat taatagtaat caattacggg 721 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 781 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 841 agtaacgcca atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc 901 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 961 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 1021 gcagtacatc tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct 1081 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt 1141 aattattttg tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg 1201 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc 1261 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga 1321 agcgcgcggc gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc 1381 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac 1441 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg 1501 tggctgcgtg aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc 1561 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc 1621 ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga 1681 gcgcggccgg gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt 1741 gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc 1801 cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc 1861 gtgcggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc 1921 cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccg 1981 gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg 2041 cgagagggcg cagggacttc ctttgtccca aatctggcgg agccgaaatc tgggaggcgc 2101 cgccgcaccc cctctagcgg gcgcgggcga agcggtgcgg cgccggcagg aaggaaatgg 2161 gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccatctc cagcctcggg 2221 gctgccgcag ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg 2281 gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct 2341 acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga 2401 tcctagtgga tcccccgggc tgcaggaatt cgatatcaca agtttgtaca aaaaagcagg 2461 ctggaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 2521 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 2581 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 2641 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 2701 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 2761 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 2821 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 2881 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 2941 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 3001 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 3061 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 3121 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 3181 caagggcggt ggaagatctg ggaattcaag gcctctcgag cctctagatt ctgcagccct 3241 atagcaccca gctttcttgt acaaagtggt gatatcaagc ttatcgatac cgtcgacctc 3301 gagggccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 3361 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 3421 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 3481 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 3541 tctatggctt ctgaggcgga aagaaccagc tggggcttaa ttaagagctc cagcttttgt 3601 tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg 3661 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 3721 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 3781 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 3841 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 3901 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 3961 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4021 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4081 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4141 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4201 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 4261 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4321 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4381 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4441 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 4501 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4561 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 4621 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 4681 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 4741 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 4801 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 4861 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 4921 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 4981 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 5041 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 5101 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 5161 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 5221 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 5281 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 5341 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 5401 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 5461 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 5521 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 5581 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 5641 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 5701 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 5761 gttccgcgca catttccccg aaaagtgcca cctaaattgt a //