LOCUS       sequence-162291-        7327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Desulfovibrio vulgaris Cas5c 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 7327)
  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 7327)
  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 Cas5c/pHMGWA
FEATURES             Location/Qualifiers
     source          1..7327
                     /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(2041..2065)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2079..2096
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2149..2167)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2163..2210
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2247..2702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2334..2353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2544..2565
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2728..2832
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2833..2901,2902..3693)
                     /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(3051..3070)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3864..4452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4353..4372
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4606..4623
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4638..4777
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4703..4722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4863..4885
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4879..5070)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5879..6961)
                     /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     6923..6942
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6962..7039)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7245..7264
                     /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 atgacacatg gggctgttaa gacatacggc atcagactgc
     1381 gggtatgggg cgactacgcc tgcttcacgc ggccggagat gaaggtggaa cgggtgtcat
     1441 acgatgtcat gcccccttcc gccgcacggg gcatccttga ggccatccac tggaagccgg
     1501 ccatcaggtg gattgtggac aggattcatg tgctgcggcc catcgtcttc gacaacgtgc
     1561 ggcgcaacga ggtcagttcg aagataccca agcccaatcc ggcaacggcc atgcgcgaca
     1621 ggaagccgct ctactttctg gtcgatgacg gcagcaaccg ccagcaacgg gcggcgaccc
     1681 tgctgcgcaa cgtcgactat gtcatcgaag cccacttcga actgacagac aaggccggag
     1741 cggaagacaa cgcgggcaag catctggaca tcttcaggcg tcgtgcgcgc gccgggcagt
     1801 cgttccagca gccatgcctc ggctgccgcg agtttccggc gtcgttcgaa ctgctggagg
     1861 gggatgtgcc gctctcgtgc tacgcgggcg agaagcgcga ccttggctac atgctgctcg
     1921 acatcgactt cgaacgcgac atgacgccgc tgttcttcaa ggccgtcatg gaagacggag
     1981 tgataacgcc cccgtcgcgc acgtcaccgg aggtgagggc atgaaagggt gggcgcgccg
     2041 acccagcttt cttgtacaaa gtggtgatgt acctcgagca ccaccaccac caccactgag
     2101 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2161 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2221 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2461 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2521 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2581 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2641 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2701 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2761 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2821 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     2881 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2941 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3001 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3061 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3121 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3181 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3241 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     3301 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     3361 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     3421 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     3481 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     3541 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     3601 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     3661 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     3721 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     3781 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     3841 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3901 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3961 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4021 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4081 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4141 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4201 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4261 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4321 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4381 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4441 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4501 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     4561 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     4621 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4681 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     4741 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     4801 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4861 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     4921 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     4981 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5041 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5101 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5161 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5221 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5281 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5341 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5401 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5461 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     5521 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     5581 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     5641 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     5701 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     5761 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5821 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5881 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5941 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     6001 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     6061 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     6121 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     6181 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6241 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6301 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6361 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6421 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6481 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6541 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6601 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6661 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6721 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6781 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6841 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6901 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6961 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     7021 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     7081 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     7141 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7201 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7261 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7321 aggatcg
//
