LOCUS       sequence-234058-        6992 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 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 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..6992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(join(2690..2695,2696..2712,2713..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             join(2936..3004,3005..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             join(6922..6992,1..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
//
