LOCUS       sequence-228507-        6995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wildtype V-D Cas1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6995)
  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 6995)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2018.12.015"; journalName: "Mol Cell"; date: 
            "2019-02-21- 21"; volume: "73"; issue: "4"; pages: "727-737.e3"
COMMENT     Sequence Label: pET-3a-His-MBP-TEV-VD_Cas1_WT
FEATURES             Location/Qualifiers
     source          1..6995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     273..292
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     526..543
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    558..700
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(626..645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     786..808
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(802..993)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2451..2470
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2554..2573
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2554..2572
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2661..2683
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2703..2732
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2742..3842
                     /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"
     primer_bind     3816..3839
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3897..3917
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5075..5093)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5089..5136
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5502..5507,5508..5524,5525..5530))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5557..5575)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5643..5747
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5748..5816,5817..6608)
                     /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(5966..5985)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6779..6995,1..372)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
       61 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      121 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      181 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      241 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      301 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      361 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      421 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      481 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      541 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      601 caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      661 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
      721 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
      781 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
      841 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
      901 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
      961 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     1021 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     1081 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     1141 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     1201 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     1261 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     1321 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     1381 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     1441 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     1501 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     1561 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     1621 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
     1681 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     1741 gcggcataaa tcgccgtgac gatcagcggt ccagtgatcg aagttaggct ggtaagagcc
     1801 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     1861 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     1921 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     1981 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     2041 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     2101 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     2161 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     2221 ctgactgggt tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc
     2281 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     2341 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     2401 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     2461 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     2521 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
     2581 aacggtttcc ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg
     2641 actcgagttc tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt
     2701 ctcaccatca ccatcaccat caccatcacc atggttcttc tatgaaaatc gaagaaggta
     2761 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
     2821 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
     2881 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
     2941 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
     3001 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
     3061 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
     3121 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
     3181 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
     3241 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
     3301 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     3361 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     3421 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     3481 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     3541 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     3601 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     3661 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     3721 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     3781 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     3841 ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa
     3901 acctgtactt ccaatccaat gcaatggcac atcgtattcc gctgtggctg ccgtatctga
     3961 gcgaagttaa tgttagcgca agcggtgtta tccactatgt ttataaaggt ggtgaagata
     4021 gcgtgcgcct gaaagatatt tcaagcatta tgatttatgg cgaaaccgat gccagcttta
     4081 gctttaaaac ctttgaaaaa atcagccgtg caggcattcc ggtgatttat acccgtcgta
     4141 atattggtag cccgatgatt attcatggtg gtctgcgtta tgatgcagtt gataccgtta
     4201 ccgcacagat tctggcacgt gaaaataaga aaaaacagtg ccatattgca cgccagctgc
     4261 tgaaagcaaa aatgaatagc atgaaacatc tggcaccggt ttttgaactg ccggataatg
     4321 caaacattaa tgaactgcgc aacatcgaag caaaacacgc caaaaaatac tggcgtgagt
     4381 attataccta tatcggtcat ccggaatgga cccgtcgcgg tgaagaaacc ggtaaaaatc
     4441 cggttagcga taccctgaat ctgaccagca aatttatctg tggcattatt ctgcgttgga
     4501 ccctgtatca tcatctgagc ccgagccatg gttattttca tgttatgacc gattatcaga
     4561 gcctggtgta tgacctgatt gaaccgtatc gtgcactgtt tgatattgaa ctgattcgcc
     4621 tgtttaaaga taacgcccat ctggaagaaa attggcctgg tatggcaatt aacctgatga
     4681 aaaacaagct gaacaaaaaa tgctattgtc cgctgacacg tcaggttgtt acctatcatg
     4741 aactgaccca tggtattgtt ctgagcctga aaagctatct gattggtcgt cagcgcaaat
     4801 ttcatattcc gttaccgggt agtccgaatg gtggtcgtcc gagcaaagtt gaatttaaac
     4861 tgtatgcacg taacgcaggc aaaaccgatt tttggaaagt tgcaaaagaa gtggccgatc
     4921 tggattttgc atgtctgagc gttggttaat aacattggaa gtggataacg gatccgcgat
     4981 cgcggcgcgc cacctggtgg ccggccggta ccacgcgtgc gcgctgatcc ggctgctaac
     5041 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     5101 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     5161 tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc caagtagcga
     5221 agcgagcagg actgggcggc ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca
     5281 tagaaattgc atcaacgcat atagcgctag cagcacgcca tagtgactgg cgatgctgtc
     5341 ggaatggacg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
     5401 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
     5461 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
     5521 aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     5581 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5641 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5701 gacaataacc ctgataaatg cttcaataac attgaaaaag gaagagtatg agtattcaac
     5761 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5821 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5881 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5941 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     6001 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6061 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6121 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6181 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6241 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     6301 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6361 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6421 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6481 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6541 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6601 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6661 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6721 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6781 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6841 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6901 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     6961 agaactctgt agcaccgcct acatacctcg ctctg
//
