LOCUS Exported 8353 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses FANCD2.. ACCESSION . VERSION . KEYWORDS pCR1317 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8353) AUTHORS Richardson CD, Kazane KR, Feng SJ, Zelin E, Bray NL, Schafer AJ, Floor SN, Corn JE TITLE CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. JOURNAL Nat Genet. 2018 Aug;50(8):1132-1139. doi: 10.1038/s41588-018-0174-0. Epub 2018 Jul 27. PUBMED 30054595 REFERENCE 2 (bases 1 to 8353) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8353 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 70..87 /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" regulatory 883..892 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" primer_bind 1841..1857 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 1841..1857 /label=pBluescriptSK /note="For pBluescript vector" repeat_region 2126..2160 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(2651..2667) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2651..2667) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(2664..2686) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 2675..2691 /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(2699..2729) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2744..2765 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2882..2899) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3053..3641) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3133..3152) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3812..4672) /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 4435..4454 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4673..4777) /gene="bla" /label=AmpR promoter rep_origin complement(4803..5258) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4940..4961) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 5152..5171 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5385..5407 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 5399..5416 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 5400..5416 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 5546..5608 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" polyA_signal 6387..6521 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6424..6443) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6478..6497 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" misc_feature 6601..7189 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" CDS 6707..6718 /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind 7116..7136 /label=WPRE-R /note="WPRE, reverse primer" CDS complement(7214..8008) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(7325..7344) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 7935..7954 /label=Neo-R /note="Neomycin resistance gene, reverse primer" misc_feature 8021..254 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(8194..8213) /label=IRES-F /note="IRES internal ribosome entry site, forward primer" ORIGIN 1 ctacagacgt tgtttgtctt caagaagctt ccagaggaac tgcttccttc acgacattca 61 acagaccttg cattcctttg gcgagagggg aaagacccct aggaatgctc gtcaagaaga 121 cagggccagg tttccgggcc ctcacattgc caaaagacgg caatatggtg gaaaataaca 181 tatagacaaa cgcacaccgg ccttattcca agcggcttcg gccagtaacg ttaggggggg 241 gggagggaga ggggcgcggc cgcggatccg atttaaattc gataggctca taggtcgagg 301 gcgctcagta gccccctaac cagctggaga agtcgagtag ctcgcgctcc gcaggactca 361 gcgcgccttc gcagccgctg tcgtccgacg agtaggcgga acgcggggag ccgggctccg 421 agctgccccc gcggcccggg gacgaagaag cgcgggaggg cgaggcggcg accggggtgg 481 tccctggcgg cccgcggggc gcagacggcc gcacggcctg cggcctcagc cctcccgcca 541 gcgcgttgcg cacggcgtcg tgctcggcca gcaggcgctg cagcgcgcgg atgtactcca 601 cggctgagcg cagcgtctcc accttgctca gcttcttgct ggcgccgccg tgcggcacgt 661 gctgccgcag cgcctggaag cccaagttca ccagcttcac gcggttgcgc tcgcgctcat 721 tgcgccgcgc tacggccgct gcgccgcctc cggtctctgc ggtggccggt cgccgccgcc 781 ggctgcagcg caacagttcc ggggacgcgg gtctccgccg ggcagcgcag ccgacaggga 841 cggggggcgc agggggcgcg gacctgggca gtgtgccgcc gtccatggtg gctagctcta 901 gagtaggcac cggtcgtggc ggccgacctg caggttcact aaaccgaatg ttcacagaga 961 ctactgcact tatatatggt tctcccccac cctggggaaa aaggtggagc cagtacacca 1021 catcactttc ccagtttacc caagccccac cttctctagg caccagttca attgcccacc 1081 cctcccccca acttctcagg gactgtgggc catgtgctct ctgcccactg aggggcactc 1141 agccctcaag catgctcttc tccactagtc acccctattg accttatgta tgtgccaata 1201 atgggaaaaa cccattgact caccccctat tgaccttttg tactgggcaa aacccaatgg 1261 aaagtcccta ttgactcagt gtacttggct ccaatgggac tttcctgttg attcacccct 1321 attgacctta tgtactgggc aaaacccatt ggaaagtccc taatgactca gtatacatgt 1381 cagtaatggg aaaaacccat tggcttacct cccattgacc ttatgtactg ggcaaaaccc 1441 attggaaagt ccctattgac tcaatgtact tggctccaat gggactttcc tgttgactca 1501 ccccctattg accttatgta ctgggcaaaa cccaatggaa agtccctatt gagtcagtgt 1561 acttggctcc aatgggtttt tcccattgac tcctgcaggc cttggaggcc ttttccccgt 1621 atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc 1681 cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg 1741 gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga 1801 cgtaattaca tccctggggg ctttgggggg gggctgtccc tctagaacta gtggatctat 1861 aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa 1921 ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc 1981 acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg 2041 gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga 2101 aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg 2161 ttaatctagc tgcatcagga tcatatcgtc gggtcttttt tccggctcag tcatcgccca 2221 agctggcgct atctgggcat cggggaggaa gaagcccgtg ccttttcccg cgaggttgaa 2281 gcggcatgga aagagtttgc cgaggatgac tgctgctgca ttgacgttga gcgaaaacgc 2341 acgtttacca tgatgattcg ggaaggtgtg gccatgcacg cctttaacgg tgaactgttc 2401 gttcaggcca cctgggatac cagttcgtcg cggcttttcc ggacacagtt ccggatggtc 2461 agcccgaagc gcatcagcaa cccgaacaat accggcgaca gccggaactg ccgtgccggt 2521 gtgcagatta atgacagcgg tgcggcgctg ggatattacg tcagcgagga cgggtatcct 2581 ggctggatgc cgcagaaatg gacatggata ccccgtgagt tacccggcgg gcgcgcttgg 2641 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 2701 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 2761 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 2821 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 2881 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2941 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 3001 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 3061 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 3121 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 3181 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 3241 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 3301 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 3361 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 3421 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 3481 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 3541 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3601 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3661 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 3721 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 3781 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3841 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3901 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 3961 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 4021 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 4081 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 4141 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 4201 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 4261 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 4321 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 4381 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 4441 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 4501 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 4561 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4621 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4681 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4741 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4801 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 4861 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 4921 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 4981 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 5041 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 5101 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 5161 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 5221 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 5281 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 5341 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 5401 taaaacgacg gccagtgagc gcgcctcgtt cattcacgtt tttgaacccg tggaggacgg 5461 gcagactcgc ggtgcaaatg tgttttacag cgtgatggag cagatgaaga tgctcgacac 5521 gctgcagaac acgcagctag attaacccta gaaagataat catattgtga cgtacgttaa 5581 agataatcat gtgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg aggtttattt 5641 attaatttga atagatatta agttttatta tatttacact tacatactaa taataaattc 5701 aacaaacaat ttatttatgt ttatttattt attaaaaaaa acaaaaactc aaaatttctt 5761 ctataaagta acaaaacttt tatcgaattc tttttccccg tatcccccca ggtgtctgca 5821 ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc 5881 ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg 5941 agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg 6001 gctttggggg ggggctgtcc ccgtgagctc cattggtatg gctttttccc cgtatccccc 6061 caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga tcccgtgcca 6121 ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc 6181 ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag ggacgtaatt 6241 acatccctgg gggctttggg ggggggctgt ccctctagag gatcataatc agccatacca 6301 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac 6361 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat 6421 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 6481 gtttgtccaa actcatcaat gtatcttatc atgtctggat cactagtatc gatatctcga 6541 ctcactacct ccctttaaac gatcgacggc cacgaagtgc ttagccagaa ttaattccag 6601 gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa 6661 gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag 6721 ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccatg 6781 gaaaggacgt cagcttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc 6841 ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga 6901 aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg 6961 cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat 7021 aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa 7081 tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt 7141 taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgattg ttccagacgc 7201 gaactctagc gactcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat 7261 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt 7321 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc 7381 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat 7441 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca 7501 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 7561 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 7621 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 7681 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 7741 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgcggcca 7801 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct 7861 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 7921 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 7981 ctgcgtgcaa tccatcttgt tcaatcattc cggacgccat ggttgtggcc atattatcat 8041 cgtgtttttc aaaggaaaac cacgtccccg tggttcgggg ggcctagacg tttttttaac 8101 ctcgactaaa cacatgtaaa gcatgtgcac cgaggcccca gatcagatcc catacaatgg 8161 ggtaccttct gggcatcctt cagccccttg ttgaatacgc ttgaggagag ccatttgact 8221 ctttccacaa ctatccaact cacaacgtgg cactggggtt gtgccgcctt tgcaggtgta 8281 tcttatacac gtggcttttg gccgcagagg cacctgtcgc caggtggggg gttccgctgc 8341 ctgcaaaggg tcg //