LOCUS       sequence-161819-        7516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Desulfovibrio vulgaris Cas7 in E. coli with TEV-cleavable 
            N-terminal His-MBP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7516)
  AUTHORS   Hochstrasser ML, Taylor DW, Kornfeld JE, Nogales E, Doudna JA
  TITLE     DNA Targeting by a Minimal CRISPR RNA-Guided Cascade.
  JOURNAL   Mol Cell. 2016 Sep 1;63(5):840-51. doi: 
            10.1016/j.molcel.2016.07.027.
  PUBMED    27588603
REFERENCE   2  (bases 1 to 7516)
  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.2016.07"; journalName: "Mol Cell"; date: 
            "2016-09-1- 1"; volume: "63"; issue: "5"; pages: "840-51"
COMMENT     Sequence Label: D. vulgaris Cas7/pHMGWA
FEATURES             Location/Qualifiers
     source          1..7516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    46..70
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             85..107
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             126..143
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             150..1247
                     /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="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1104..1113
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1221..1244
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     protein_bind    1266..1290
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1311..1331
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(2230..2254)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2268..2285
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2338..2356)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2352..2399
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2436..2891
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2523..2542)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2733..2754
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2917..3021
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3022..3090,3091..3882)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3240..3259)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4053..4641
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4542..4561
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4795..4812
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4827..4966
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4892..4911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5052..5074
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5068..5259)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6068..7150)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7112..7131
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7151..7228)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7434..7453
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatccagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
       61 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgggca
      121 gcagccatca tcatcatcat cacggtacca aaactgaaga aggtaaactg gtaatctgga
      181 ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata
      241 ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg
      301 cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg
      361 ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc
      421 cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg
      481 aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag
      541 agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc
      601 tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt
      661 atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg
      721 gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact
      781 ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg
      841 catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca
      901 agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc
      961 cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg
     1021 aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt
     1081 tggcgaaaga tccacgtatt gccgccacca tggaaaacgc ccagaaaggt gaaatcatgc
     1141 cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg
     1201 ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactggt accggatctt
     1261 acatcacaag tttgtacaaa aaagcaggct ccgcggccgc cgtactcgag gaaaacctgt
     1321 attttcaggg ctccttcacc atgaccgcca ttgccaacag atacgagttc gtgctgcttt
     1381 tcgatgtgga gaacggcaac cccaacggcg accccgacgc gggcaacatg ccgcgcatcg
     1441 accccgagac cgggcatggt ctggtcaccg acgtgtgcct gaagcgcaag atacgcaacc
     1501 atgtggccct gacaaaagaa ggtgccgagc ggttcaatat ctatattcag gaaaaggcca
     1561 ttctcaacga aacccacgaa cgcgcctaca cggcctgcga cctgaaaccc gaacccaaga
     1621 aactgcccaa gaaggtcgaa gacgccaaga gggtcaccga ctggatgtgc acgaacttct
     1681 acgacatccg cacgttcggc gccgtcatga ccacagaggt caactgcggg caggtgcgcg
     1741 gccccgtgca gatggccttt gccagaagtg tggaacccgt cgtgccgcaa gaggtgagca
     1801 tcacccgcat ggcagtcacc acgaaggcag aggccgaaaa gcagcaggga gacaaccgca
     1861 ccatgggtcg caagcacatc gtgccctacg gcctgtatgt ggcgcacggc ttcatctccg
     1921 caccgctggc agagaagaca ggcttctccg atgaagacct gacgctgttc tgggacgccc
     1981 tcgtcaacat gttcgagcac gaccgttcgg cggcacgcgg cctcatgagc agccgcaaac
     2041 tcatcgtctt caagcaccag aacaggctcg gcaacgcccc cgcccacaag ttgttcgacc
     2101 tcgtgaaggt cagccgtgca gagggttcgt ccggccccgc ccggtcgttt gccgactatg
     2161 cggtgacagt ggggcaggcc cccgaaggcg tagaggtcaa agagatgctg tagaagggtg
     2221 ggcgcgccga cccagctttc ttgtacaaag tggtgatgta cctcgagcac caccaccacc
     2281 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     2341 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     2401 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     2461 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2521 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2581 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2641 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2701 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2761 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2821 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2881 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2941 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3001 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3061 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3121 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3181 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3241 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3301 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3361 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3421 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3481 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3541 gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact
     3601 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3661 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3721 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3781 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3841 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3901 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3961 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4021 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4081 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4141 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4201 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4261 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4321 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4381 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4441 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4501 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4561 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4621 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4681 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4741 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4801 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4861 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4921 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4981 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5041 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     5101 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     5161 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     5221 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     5281 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     5341 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     5401 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     5461 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     5521 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     5581 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     5641 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     5701 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     5761 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     5821 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     5881 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     5941 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     6001 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     6061 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     6121 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     6181 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     6241 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     6301 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     6361 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     6421 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     6481 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     6541 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     6601 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     6661 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     6721 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     6781 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6841 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6901 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6961 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     7021 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     7081 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     7141 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     7201 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     7261 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
     7321 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     7381 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     7441 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     7501 ccggcgtaga ggatcg
//
