LOCUS Exported 7388 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Base plasmid for cloning gene duplication alleles with eGFP reporter.. ACCESSION . VERSION . KEYWORDS p2T-CAG-MCS-P2A-GFP-PuroR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7388) AUTHORS Shen MW, Arbab M, Hsu JY, Worstell D, Culbertson SJ, Krabbe O, Cassa CA, Liu DR, Gifford DK, Sherwood RI TITLE Predictable and precise template-free CRISPR editing of pathogenic variants. JOURNAL Nature. 2018 Nov;563(7733):646-651. doi: 10.1038/s41586-018-0686-x. Epub 2018 Nov 7. PUBMED 30405244 REFERENCE 2 (bases 1 to 7388) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7388 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" primer_bind 1600..1622 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1684..1703 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 1772..1828 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" regulatory 1838..1847 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1844..2563 /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 1844..2560 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1889..1910) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2150..2169) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2497..2518 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2608..2742 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2632..2651) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2686..2705 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(2972..3571) /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(3055..3075) /label=Puro-F /note="Puromycin resistance gene, forward primer" primer_bind 3552..3571 /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" promoter complement(3664..3993) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3678..3813 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(3732..3751) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 3978..3998 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" primer_bind complement(4264..4284) /label=T3 /note="T3 promoter, forward primer" promoter complement(4264..4282) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(4303..4319) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4303..4319) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4316..4338) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4327..4343 /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(4351..4381) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4396..4417 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4534..4551) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4705..5293) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4785..4804) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5464..6324) /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 6087..6106 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6325..6429) /gene="bla" /label=AmpR promoter primer_bind complement(6592..6613) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 6804..6823 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7037..7059 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7051..7068 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7052..7068 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 7078..7097 /label=T7 /note="T7 promoter, forward primer" promoter 7078..7096 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 7326..7335 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 7341..7374 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 481 tgggggcggg gggggggggg gggcgcgcgc cggggggggg gggggggggg gggggggggg 541 gggggggggg gggggggggg ggggcggcgg ccaatcagag cggcgcgctc cgaaagtttc 601 cttttatggc ggggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggggg 661 gggtcgctgc gcgctgcctt cgccccgtgc cccgccccgc cgccgcctcg cgccgcccgc 721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 781 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 841 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 901 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 961 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 1021 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt 1081 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc 1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 1261 ggccgccccg ggccgggggg ggctcggggg gggggcgcgg cggcccccgg ggcgccggcg 1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 1381 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 1441 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 1501 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 1561 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 1621 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc 1681 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc ctcgagcggc 1741 cgccagtgtg atggatatcg gatccgctag cgctactaac ttcagcctgc tgaagcaggc 1801 tggagacgtg gaggagaacc ctggacctgg accggtcgcc accatggtga gcaagggcga 1861 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 1921 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 1981 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 2041 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 2101 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 2161 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 2221 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 2281 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 2341 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 2401 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 2461 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 2521 cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc gccaccgcgg 2581 tggagctcga attaattcat cgatgatgat ccagacatga taagatacat tgatgagttt 2641 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct 2701 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt 2761 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc 2821 tacaaatgtg gtatggctga ttatgatcct ctagagtcgg tgggcctcgg gggcgggtgc 2881 ggggtcggcg gggccgcccc gggtggcttc ggtcggagcc atggggtcgt gcgctccttt 2941 cggtcgggcg ctgcgggtcg tggggcgggc gtcaggcacc gggcttgcgg gtcatgcacc 3001 aggtgcgcgg tccttcgggc acctcgacgt cggcggtgac ggtgaagccg agccgctcgt 3061 agaaggggag gttgcggggc gcggaggtct ccaggaaggc gggcaccccg gcgcgctcgg 3121 ccgcctccac tccggggagc acgacggcgc tgcccagacc cttgccctgg tggtcgggcg 3181 agacgccgac ggtggccagg aaccacgcgg gctccttggg ccggtgcggc gccaggaggc 3241 cttccatctg ttgctgcgcg gccagccggg aaccgctcaa ctcggccatg cgcgggccga 3301 tctcggcgaa caccgccccc gcttcgacgc tctccggcgt ggtccagacc gccaccgcgg 3361 cgccgtcgtc cgcgacccac accttgccga tgtcgagccc gacgcgcgtg aggaagagtt 3421 cttgcagctc ggtgacccgc tcgatgtggc ggtccgggtc gacggtgtgg cgcgtggcgg 3481 ggtagtcggc gaacgcggcg gcgagggtgc gtacggcccg ggggacgtcg tcgcgggtgg 3541 cgaggcgcac cgtgggcttg tactcggtca tggaaggtcg tctccttgtg aggggtcagg 3601 ggcgtgggtc aggggatggt ggcggcaccg gtcgtggcgg ccgacctgca ggcatgcaag 3661 ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga 3721 ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga 3781 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt 3841 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt 3901 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga 3961 gcctggggac tttccacacc ctaactgaca cacattccac agaattcaag tgatctccaa 4021 aaaataagta ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct 4081 aaaaaaatgt aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta 4141 aaaatcccca aaaataatac ttaagtacag taatcaagta aaattactca agtactttac 4201 acctctggtt cttgaccccc taccttcagc aagcccagca gatccgagct ccagcttttg 4261 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt 4321 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 4381 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 4441 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 4501 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 4561 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 4621 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4681 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4741 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4801 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4861 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 4921 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4981 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 5041 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 5101 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 5161 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 5221 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 5281 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 5341 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 5401 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 5461 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 5521 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 5581 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 5641 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 5701 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 5761 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 5821 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 5881 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 5941 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 6001 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 6061 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 6121 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 6181 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 6241 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 6301 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 6361 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 6421 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta 6481 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc 6541 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg 6601 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 6661 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc 6721 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 6781 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgcgg 6841 gggggtgggg gggtcacgcg gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta 6901 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 6961 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 7021 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa 7081 tacgactcac tatagggcga attgggtacc ggcatatggt tcttgacaga ggtgtaaaaa 7141 gtactcaaaa attttactca agtgaaagta caagtactta gggaaaattt tactcaatta 7201 aaagtaaaag tatctggcta gaatcttact tgagtaaaag taaaaaagta ctccattaaa 7261 attgtacttg agtattaagg aagtaaaagt aaaagcaaga aagatcgatc tcgaaggatc 7321 tggaggccac catggtgtcg ataacttcgt atagcataca ttatacgaag ttatcgataa 7381 gcttgctc //