LOCUS Exported 6918 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses type I-F P. aeruginosa cascade (PaeCascade) proteins Csy1 and Csy2 in mammalian cells. ACCESSION . VERSION . KEYWORDS pCsy1-Csy2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6918) AUTHORS Chen Y, Liu J, Zhi S, Zheng Q, Ma W, Huang J, Liu Y, Liu D, Liang P, Songyang Z TITLE Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells. JOURNAL Nat Commun. 2020 Jun 19;11(1):3136. doi: 10.1038/s41467-020-16880-8. PUBMED 32561716 REFERENCE 2 (bases 1 to 6918) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6918 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" enhancer 154..533 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 534..737 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 688..708 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 767..787 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1772..1852 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" primer_bind complement(1880..1897) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1886..2093 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2100..2599 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind complement(2135..2158) /label=MSCV-rev /note="Murine stem cell virus, reverse primer" primer_bind 2532..2551 /label=mPGK-F /note="Mouse PGK promoter, forward primer" CDS 2628..2648 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3957..4022 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 4057..4178 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4068..4087) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 4122..4141 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 4181..4321 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4181..4310 /label=AAV2 ITR rep_origin 4396..4851 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4483..4502) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4693..4714 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4868..4887) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4987..5009 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5047..5065) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5133..5237 /gene="bla" /label=AmpR promoter CDS 5238..6098 /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(5456..5475) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6269..6857 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6758..6777 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 721 tctatataag cagagctctc tggctaacta ccggcgcgcc accatgccta agaagaagag 781 aaaagtgagc gtgactgatc ccgaggcgct gctgttgctg ccacgcctgt ccatccagaa 841 cgccaacgcc atttccagcc ccttgacctg gggtttccct tcgcccggcg ctttcaccgg 901 attcgtccat gccttgcagc gtcgagtcgg aatctccctc gatatcgaac tggacggtgt 961 cggcatcgtc tgtcaccgct tcgaagcgca gatatcgcaa ccggccggca agcgcaccaa 1021 ggtcttcaac ctgacccgca atccgctgaa ccgcgacggc tccaccgcag ccatcgtcga 1081 agaaggtcgc gcccatctgg aggtcagtct gctgctcgga gtgcatggcg atggtctgga 1141 cgatcacccc gcacaggaaa tcgccaggca ggtacaggag caggctggcg ccatgcgcct 1201 cgccggtggc agcatcctgc cctggtgcaa tgagcgcttc ccggctccga acgccgaact 1261 gctgatgctg ggtggcagcg acgaacagcg gcgcaagaac cagcgccggc ttacccgccg 1321 cctgctcccc ggcttcgccc tggtgagtcg cgaggcgttg ttgcaacaac atctggaaac 1381 cttgcgcacc acgctccccg aagccaccac gctcgatgca ctgctcgatc tttgtcgaat 1441 caacttcgag cctcctgcaa cctcgtccga agaagaagca tcgccgcccg atgcagcctg 1501 gcaagtgcgc gacaagcccg gctggctggt gccgatcccg gctggctaca acgccctgtc 1561 acccctgtac ctccccggcg aagtgcgaaa tgcccgtgat agagaaacac cgctgcgctt 1621 cgtcgaaaac ctcttcggtc tgggcgaatg gctcagcccc catcgcgtcg ccgcgttatc 1681 ggacctgctt tggtatcacc acgccgagcc tgacaaaggg ctctaccgct ggagcacacc 1741 ccgtttcgtc gaacacgcca tcgcaggatc ctacccatac gatgttccag attacgctta 1801 cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg cttaagaatt 1861 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc 1921 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 1981 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 2041 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggaggtaccg 2101 ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg 2161 cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg 2221 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag 2281 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg 2341 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg 2401 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg 2461 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc 2521 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc 2581 ctcatctccg ggcctttcga cctgcatccc gcggccgcgc caccatgccc aagaagaaga 2641 ggaaagtcac ctctcccctc ccaacgccta cgtggcagga gcttcgccag ttcatcgaat 2701 ccttcatcca ggagcgcctc cagggcaagc tggacaaact ccaacccgac gaagacgaca 2761 agcgccagac attgctggcc acccaccggc gggaggcctg gctggccgat gccgcccggc 2821 gggttggcca gttgcagttg gtgacccaca cgctcaagcc gatccatccc gacgcccgcg 2881 gcagcaacct gcacagcctg ccgcaagcac ccggccaacc gggcctcgcc ggttcccatg 2941 agctaggtga caggctggtc agcgatgtgg tgggcaatgc cgcggcgctg gacgtattca 3001 agtttctcag tctccagtat cagggtaaaa atcttctgaa ctggctgaca gaagacagtg 3061 ccgaggcatt acaggcgctg tccgataacg ccgaacaggc tcgcgaatgg cggcaagcgt 3121 tcatcggcat cacgaccgtc aaaggcgctc ccgcgtccca cagcctggcc aagcagttgt 3181 actttcccct gcccggttcc ggctaccacc tgctagcacc gctgtttccc accagtctgg 3241 tgcatcacgt ccacgctctg ctccgcgaag cgcgcttcgg cgacgcggcc aaggcggcac 3301 gcgaagcgcg cagccgccag gagtcatggc cccacggatt cagcgagtac cccaacctgg 3361 cgatccagaa gttcggcggt accaagccgc agaacatcag tcagttgaac aacgagcgcc 3421 gtggcgagaa ctggttgctg ccatcgcttc cgccgaactg gcaaaggcag aatgtaaacg 3481 cgccgatgcg gcactcttcg gtcttcgagc atgacttcgg aagaactcct gaagtatccc 3541 gcctaacccg taccctgcag cgttttcttg ccaagacagt ccataacaac ctcgccatcc 3601 gccagcggcg tgcccaattg gtggcgcaaa tctgcgacga agccctgcaa tacgccgccc 3661 gtctgcgcga actggagccc ggctggagcg caacccccgg gtgccaactg catgacgcgg 3721 agcagctctg gctcgatccg ttgcgcgcac agaccgatga gacgttcttg cagcgccgac 3781 tacgaggtga ctggcctgcc gaggtcggaa atcgcttcgc caactggctg aaccgggcgg 3841 tcagcagcga cagtcagata ctgggtagcc cggaagccgc ccaatggagc caggagctga 3901 gcaaggaact gacgatgttc aaggagatac tcgaagatga gcgtgaccaa gcttgcgact 3961 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 4021 agtaagggcc cgtgtaggct agtcgagcag acatgataag atacattgat gagtttggac 4081 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg 4141 ctttatttgt aaccattata agctgcaata aacaagttgc aggaacccct agtgatggag 4201 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 4261 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag 4321 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt 4381 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 4441 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc 4501 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 4561 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg 4621 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 4681 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct 4741 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga 4801 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca 4861 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 4921 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 4981 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 5041 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 5101 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 5161 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 5221 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 5281 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5341 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5401 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 5461 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 5521 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 5581 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 5641 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 5701 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5761 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 5821 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5881 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5941 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc 6001 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 6061 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 6121 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 6181 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 6241 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6301 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 6361 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 6421 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 6481 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 6541 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 6601 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 6661 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 6721 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 6781 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6841 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 6901 ggccttttgc tcacatgt //