LOCUS       sequence-228509-        7094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses wildtype V-C Cas1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7094)
  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 7094)
  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-VC_Cas1_WT
FEATURES             Location/Qualifiers
     source          1..7094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1487..1506
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1590..1609
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1590..1608
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1697..1719
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1739..1768
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1778..2878
                     /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     2852..2875
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2933..2953
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4210..4228)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4224..4271
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4637..4642,4643..4659,4660..4665))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4692..4710)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4778..4882
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4883..4951,4952..5743)
                     /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(5101..5120)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5914..6502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6403..6422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6656..6673
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6688..6830
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6756..6775)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6916..6938
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(6932..7094,1..29))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
       61 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
      121 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      181 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      241 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
      301 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
      361 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
      421 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
      481 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
      541 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
      601 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
      661 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
      721 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
      781 cataaatcgc cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcga
      841 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
      901 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
      961 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     1021 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     1081 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     1141 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     1201 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     1261 ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc cttatgcgac tcctgcatta
     1321 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     1381 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     1441 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     1501 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     1561 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg
     1621 gtttccctct agtgccggct ccggagagct ctttaattaa gcggccgccc tgcaggactc
     1681 gagttctaga aataattttg tttaacttta agaaggagat atacatatga aatcttctca
     1741 ccatcaccat caccatcacc atcaccatgg ttcttctatg aaaatcgaag aaggtaaact
     1801 ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt
     1861 cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt
     1921 cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt
     1981 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga
     2041 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc
     2101 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa
     2161 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct
     2221 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta
     2281 tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg
     2341 cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga
     2401 caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa
     2461 cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact
     2521 gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa
     2581 cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga
     2641 tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta
     2701 cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg
     2761 tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt
     2821 gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa
     2881 ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg aggaaaacct
     2941 gtacttccaa tccaatgcaa tggatcaggg taatcagacc attgaaaacc agaccattaa
     3001 ttgtgatcag cacccggatt ttctgtggac ctggaaaagc aataaacgtg gtagccgtgt
     3061 tagcgtttgg ctgccgtatt ttagccaggc caaaaaaatc cctcgtagca aaaaatggtc
     3121 agtggcatat aatggtggca gcattgaatt cgatctgaaa gaaaccgatc tgatcatgtt
     3181 ttatggtgca accggtgaac tgccgctgga atttctggat gatgcaagca aaaatggtgt
     3241 gatgattctg attcatcgtc gtaatgttct gcagccgtat gttttttatc cgagcgttat
     3301 tggtgatgaa gaagatattc tgaccaagca gattcagttt cgtaccaatg aacgtaaacg
     3361 tctgtatatt gcaaagaccc tgatcaaaaa acgcctggaa aatatgggta gcaccattcc
     3421 gattagcgca ccgctgctgc gtcagctgag tgcagcaaaa agcattgatg aagttcgtgc
     3481 aattgaagca aataccaccg cacgttattg gaacaaatgg tatgaaaacc tgaacatcga
     3541 aaccacgcgt cgtaaagatc atccgattaa tagcgcactg gatgcaggta gcaaatttat
     3601 ctatggtgtt attctgcgtt ggctggtgtt tcatcgtttt agcccgaatc atggctttat
     3661 gcatcagccg accagctatc cgagcctggt ttatgatctg atggaaccgt ttcgctatat
     3721 gattgaaaat gtttgtagcg cagcatggaa acgtggtgaa cgtgaaaata gcaaaattgt
     3781 tgcactgagc ctgagctttc tgaaagagga actggataaa ccgtgttatg ttccggcaac
     3841 acgtcagtat gttcgcaaaa aaaacctgct gcatggtgca gttctggcac tgcgtagcta
     3901 tctgattggt gatatgcgta aactggtttt tccgagcgaa ggtgttccga atggtggtcg
     3961 tccgattaaa gcaagctata aactgcctgg tagcatgtat gatgttggtc gtaaaccgcc
     4021 tgaaatcaaa cagaaagatg aaatctgctt tgacgaagtg agccaagaag aaagcgaaga
     4081 gtaataacat tggaagtgga taacggatcc gcgatcgcgg cgcgccacct ggtggccggc
     4141 cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     4201 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     4261 ggggtttttt gctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc
     4321 atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca
     4381 aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc
     4441 gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc ccgcaagagg
     4501 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     4561 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     4621 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     4681 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4741 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4801 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4861 ataacattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4921 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4981 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5041 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5101 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     5161 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5221 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5281 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5341 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5401 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     5461 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5521 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5581 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5641 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5701 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5761 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5821 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5881 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5941 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6001 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6061 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6121 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6181 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6241 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6301 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6361 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6421 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6481 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6541 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6601 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6661 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     6721 cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6781 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     6841 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     6901 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     6961 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     7021 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     7081 taaagcgggc catg
//
