LOCUS Exported 6992 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses wildtype V-D Cas1. ACCESSION . VERSION . KEYWORDS pET-3a-His-MBP-TEV-VD_Cas1_WT SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6992) AUTHORS Wright AV, Wang JY, Burstein D, Harrington LB, Paez-Espino D, Kyrpides NC, Iavarone AT, Banfield JF, Doudna JA TITLE A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System. JOURNAL Mol Cell. 2019 Feb 21;73(4):727-737.e3. doi: 10.1016/j.molcel.2018.12.015. Epub 2019 Jan 29. PUBMED 30709710 REFERENCE 2 (bases 1 to 6992) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6992 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1004..1027 /label=MBP-F /note="Maltose binding protein, forward primer" CDS 1085..1105 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" primer_bind complement(2263..2281) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2277..2324 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(2690..2718) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" primer_bind complement(2745..2763) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2831..2935 /gene="bla" /label=AmpR promoter CDS 2936..3796 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(3154..3173) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3967..4555 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4456..4475 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4709..4726 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4741..4880 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4806..4825) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4966..4988 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4982..5173) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" primer_bind 6631..6650 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 6734..6753 /label=T7 /note="T7 promoter, forward primer" promoter 6734..6752 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 6841..6863 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 6883..6912 /codon_start=1 /product="10xHis affinity tag" /label=10xHis /translation="HHHHHHHHHH" CDS 6922..1030 /codon_start=1 /gene="malE (mutated)" /product="maltose binding protein from E. coli" /label=MBP /note="This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol." /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" ORIGIN 1 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact 61 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc 121 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa 181 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct 241 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc 301 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg 361 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc 421 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt 481 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca 541 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc 601 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg 661 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag 721 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta 781 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc 841 gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa 901 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt 961 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga 1021 cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat 1081 cgaggaaaac ctgtacttcc aatccaatgc aatggcacat cgtattccgc tgtggctgcc 1141 gtatctgagc gaagttaatg ttagcgcaag cggtgttatc cactatgttt ataaaggtgg 1201 tgaagatagc gtgcgcctga aagatatttc aagcattatg atttatggcg aaaccgatgc 1261 cagctttagc tttaaaacct ttgaaaaaat cagccgtgca ggcattccgg tgatttatac 1321 ccgtcgtaat attggtagcc cgatgattat tcatggtggt ctgcgttatg atgcagttga 1381 taccgttacc gcacagattc tggcacgtga aaataagaaa aaacagtgcc atattgcacg 1441 ccagctgctg aaagcaaaaa tgaatagcat gaaacatctg gcaccggttt ttgaactgcc 1501 ggataatgca aacattaatg aactgcgcaa catcgaagca aaacacgcca aaaaatactg 1561 gcgtgagtat tatacctata tcggtcatcc ggaatggacc cgtcgcggtg aagaaaccgg 1621 taaaaatccg gttagcgata ccctgaatct gaccagcaaa tttatctgtg gcattattct 1681 gcgttggacc ctgtatcatc atctgagccc gagccatggt tattttcatg ttatgaccga 1741 ttatcagagc ctggtgtatg acctgattga accgtatcgt gcactgtttg atattgaact 1801 gattcgcctg tttaaagata acgcccatct ggaagaaaat tggcctggta tggcaattaa 1861 cctgatgaaa aacaagctga acaaaaaatg ctattgtccg ctgacacgtc aggttgttac 1921 ctatcatgaa ctgacccatg gtattgttct gagcctgaaa agctatctga ttggtcgtca 1981 gcgcaaattt catattccgt taccgggtag tccgaatggt ggtcgtccga gcaaagttga 2041 atttaaactg tatgcacgta acgcaggcaa aaccgatttt tggaaagttg caaaagaagt 2101 ggccgatctg gattttgcat gtctgagcgt tggttaataa cattggaagt ggataacgga 2161 tccgcgatcg cggcgcgcca cctggtggcc ggccggtacc acgcgtgcgc gctgatccgg 2221 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 2281 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 2341 tatccggata tccacaggac gggtgtggtc gccatgatcg cgtagtcgat agtggctcca 2401 agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg 2461 ggtgcgcata gaaattgcat caacgcatat agcgctagca gcacgccata gtgactggcg 2521 atgctgtcgg aatggacgat atcccgcaag aggcccggca gtaccggcat aaccaagcct 2581 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc 2641 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta 2701 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct 2761 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg 2821 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 2881 gctcatgaga caataaccct gataaatgct tcaataacat tgaaaaagga agagtatgag 2941 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3001 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 3061 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 3121 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 3181 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 3241 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 3301 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 3361 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 3421 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc 3481 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 3541 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 3601 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 3661 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 3721 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 3781 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 3841 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 3901 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 3961 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4021 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4081 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 4141 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 4201 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 4261 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 4321 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 4381 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 4441 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 4501 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 4561 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 4621 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 4681 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 4741 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact 4801 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 4861 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 4921 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 4981 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag 5041 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt 5101 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt 5161 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac 5221 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac 5281 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca 5341 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc 5401 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca 5461 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt 5521 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa 5581 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg 5641 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga 5701 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg 5761 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga 5821 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc 5881 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac 5941 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg 6001 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat 6061 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc 6121 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc 6181 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa 6241 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat 6301 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag 6361 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct 6421 caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag 6481 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc 6541 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag 6601 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac 6661 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc 6721 gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagtgcc 6781 ggctccggag agctctttaa ttaagcggcc gccctgcagg actcgagttc tagaaataat 6841 tttgtttaac tttaagaagg agatatacat atgaaatctt ctcaccatca ccatcaccat 6901 caccatcacc atggttcttc tatgaaaatc gaagaaggta aactggtaat ctggattaac 6961 ggcgataaag gctataacgg tctcgctgaa gt //