LOCUS Exported 3161 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Empty vector for cloning AsCpf1-SpCas9 fgRNA. ACCESSION . VERSION . KEYWORDS pFGRNA-AsCpf1-SpCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3161) AUTHORS Kweon J, Jang AH, Kim DE, Yang JW, Yoon M, Rim Shin H, Kim JS, Kim Y TITLE Publisher Correction: Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. JOURNAL Nat Commun. 2018 Jan 16;9(1):303. doi: 10.1038/s41467-017-02686-8. PUBMED 29335559 REFERENCE 2 (bases 1 to 3161) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3161 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind 344..362 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(400..422) /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(517..834) /codon_start=1 /gene="lacZ fragment" /product="LacZ-alpha fragment of beta-galactosidase" /label=lacZ-alpha /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDA A" primer_bind 522..541 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 735..757 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 749..766 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 750..766 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_feature 770..826 /label=MCS /note="pUC18/19 multiple cloning site" primer_bind complement(835..851) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(835..851) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(848..870) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 859..875 /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(883..913) /label=lac promoter /note="promoter for the E. coli lac operon" misc_RNA 943..1018 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(1152..1169) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1323..1911) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1403..1422) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2082..2942) /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 2705..2724 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2943..3047) /gene="bla" /label=AmpR promoter ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg taatttctac tcttgtagat ggagaccaca tttccccgaa aagtgccacc 301 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 361 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 421 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 481 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt 541 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg 601 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 661 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 721 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgcca agcttgcatg 781 cctgcaggtc gactctagag gatccccggg taccgagctc gaattcgtaa tcatgtcata 841 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 901 cataaagtgt aaagcctggg gtgcctaata gatccggtct ccgttttaga gctagaaata 961 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 1021 ttttctagat tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt 1081 gtcttcctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 1141 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 1201 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 1261 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 1321 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 1381 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 1441 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 1501 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 1561 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 1621 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 1681 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 1741 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 1801 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 1861 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 1921 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 1981 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 2041 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 2101 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 2161 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 2221 cgagatccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 2281 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 2341 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 2401 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 2461 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 2521 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 2581 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 2641 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 2701 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 2761 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 2821 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 2881 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 2941 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 3001 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 3061 aaagtgccac ctgacgtcga cgaagactca attgtcgatt agtgaacgga tctcgacggt 3121 atcgatcacg agactagcct cgagcggccg cccccttcac c //