LOCUS       sequence-255644-        9521 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses V. cholerae CAST Cas8, Cas7, and Cas6 from a T7 promoter, 
            with N-terminal His10-MBP-TEVsite tag on Cas8 for purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9521)
  AUTHORS   Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH
  TITLE     Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA 
            integration.
  JOURNAL   Nature. 2019 Jul;571(7764):219-225. doi: 10.1038/s41586-019-1323-z. 
            Epub 2019 Jun 12.
  PUBMED    31189177
REFERENCE   2  (bases 1 to 9521)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-019-1323-z"; journalName: "Nature"; date: "2019-07";
            volume: "571"; issue: "7764"; pages: "219-225"
COMMENT     Sequence Label: pSL0374 (His-MBP-Cas8_Cas7_Cas6)
FEATURES             Location/Qualifiers
     source          1..9521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2665..2684
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2768..2787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2768..2786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2875..2897
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2917..2946
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2956..4056
                     /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     4030..4053
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4111..4131
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7818..7836)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7832..7879
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8245..8250,8251..8267,8268..8273))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8300..8318)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8386..8490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8491..8559,8560..9351)
                     /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(8709..8728)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     1681 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     1741 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     1801 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     1861 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     1921 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     1981 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     2041 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     2101 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     2161 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     2221 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     2281 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     2341 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     2401 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     2461 catcggtcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt
     2521 gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag
     2581 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
     2641 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
     2701 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
     2761 gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag tgccggctcc
     2821 ggagagctct ttaattaagc ggccgccctg caggactcga gttctagaaa taattttgtt
     2881 taactttaag aaggagatat acatatgaaa tcttctcacc atcaccatca ccatcaccat
     2941 caccatggtt cttctatgaa aatcgaagaa ggtaaactgg taatctggat taacggcgat
     3001 aaaggctata acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa
     3061 gtcaccgttg agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc
     3121 gatggccctg acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc
     3181 ctgttggctg aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg
     3241 gatgccgtac gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg
     3301 ctgatttata acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg
     3361 ctggataaag aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg
     3421 tacttcacct ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc
     3481 aagtacgaca ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc
     3541 ctggttgacc tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa
     3601 gctgccttta ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac
     3661 atcgacacca gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca
     3721 tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag
     3781 ctggcaaaag agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat
     3841 aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat
     3901 ccacgtattg ccgccactat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg
     3961 cagatgtccg ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt
     4021 cagactgtcg atgaagccct gaaagacgcg cagactaatt cgagctcgaa caacaacaac
     4081 aataacaata acaacaacct cgggatcgag gaaaacctgt acttccaatc caatgcaatg
     4141 caaactctga aagaactaat cgcatccaat cctgacgact taacaactga acttaagaga
     4201 gcatttcgtc cactcacacc gcatattgca attgatggta atgaacttga cgcactgacg
     4261 atattagtca atttaaccga taagactgat gatcagaaag acctgctcga tcgagccaaa
     4321 tgcaagcaaa aacttcgaga tgaaaaatgg tgggctagct gcataaattg cgttaattac
     4381 agacaaagcc ataacccaaa attcccggat atacgttctg aaggcgtgat tcgaacccaa
     4441 gccctgggtg aattaccgag tttcctactc tcgtcctcaa aaatcccacc ataccattgg
     4501 tcatatagcc atgattcaaa atacgtcaac aaaagcgcat tcctcaccaa tgagttttgt
     4561 tgggatggtg agatctcatg tttgggtgag cttcttaaag atgcagatca cccactttgg
     4621 aatactttga aaaagttagg ttgttctcaa aaaacttgca aagcaatggc aaaacaacta
     4681 gctgatatta ctctcacgac tatcaatgtc acactcgcac caaattacct gactcaaata
     4741 tctctccccg atagtgatac atcctacatt tcactctctc ctgttgcatc gctatcgatg
     4801 caaagccact ttcatcagag gctccaagat gaaaatcggc atagcgcgat aacgcggttt
     4861 agccgaacta ccaacatggg ggtcactgcg atgacatgtg gcggcgcatt tagaatgtta
     4921 aagtctggcg ctaagttttc cagcccccct catcaccgat taaacagtaa acgaagttgg
     4981 ttgacgtcag agcatgttca gtcattaaaa cagtaccagc gcctcaacaa aagcctcata
     5041 cctgaaaact ctcggattgc actccgtaga aaatacaaaa tcgagcttca aaatatggtc
     5101 agatcttggt ttgcaatgca agaccatacc cttgattcga acatacttat ccaacatttg
     5161 aatcatgacc tatcttactt aggagccaca aaacgttttg catacgatcc agcgatgacc
     5221 aagctcttta ctgagctttt gaaacgagag ttatcaaatt caatcaataa tggtgagcaa
     5281 cacactaatg gatcgttttt agtcctaccg aatatcaggg tttgtggcgc aacagcttta
     5341 agctccccgg taacggtggg gattccatca cttacagctt tctttggctt cgttcacgca
     5401 tttgaacgga atataaatcg caccacctca tcgtttcgtg ttgaatcctt tgcgatatgc
     5461 gtccatcaac tacatgtcga gaagcgaggt ttgacagcag agtttgtgga aaaaggcgac
     5521 gggactatat ccgctcccgc gacccgggat gactggcagt gtgatgtcgt atttagcctt
     5581 attttgaaca ccaactttgc tcaacatatt gaccaagata cgttagttac atcactacca
     5641 aagcgattgg ctcggggttc agcaaaaatt gcgattgatg actttaaaca tatcaactca
     5701 ttctcgacat tagaaacagc gatcgaatct ctgccaatag aagctggtag gtggttatca
     5761 ctttacgcac agtcaaacaa taatctaagt gatctattag cagccatgac agaggaccat
     5821 cagctcatgg caagctgcgt cggttaccac ttgttagaag agcccaaaga taaaccaaac
     5881 tccctcagag gttacaaaca cgctatcgcc gagtgcatca ttggactcat taactcaatc
     5941 acctttagct cagagactga tcccaacaca atcttttggt cgctaaagaa ctatcaaaac
     6001 tacctagtgg tacagccaag gagtatcaac gatgaaacta ccgacaaatc tagcctatga
     6061 gcgctctatc gacccatcag atgtctgttt ttttgtcgtc tggcccgatg atagaaaaac
     6121 acctttaacc tacaattctc gtactctgct cgggcaaatg gaagcggcat cattagccta
     6181 tgatgtctca ggtcaaccaa taaaaagtgc caccgctgag gcgttagctc aagggaaccc
     6241 tcatcaagtt gatttctgcc acgttccata cggtgcgagt catattgaat gcagtttctc
     6301 cgtctcgttt tcttctgaac tacgtcaacc atataagtgt aactcaagca aagttaaaca
     6361 aacgctagtg caattagtcg agctctacga aacgaaaatc ggctggactg agctagcaac
     6421 ccgatatttg atgaacattt gcaacggtaa atggctgtgg aaaaataccc gtaaagccta
     6481 ttgctggaac attgtactta caccttggcc ttggaacggg gaaaaggttg gatttgaaga
     6541 tattcgtact aactacacct cacggcaaga ctttaaaaat aataaaaatt ggtctgctat
     6601 agttgaaatg atcaaaaccg cattttctag tactgatggg ctggcgatat ttgaagtcag
     6661 ggccaccttg cacttgccaa cgaatgctat ggtgcggcca agccaagttt tcacagaaaa
     6721 agaaagtggc agtaaaagta aatctaaaac tcaaaacagt cgagtttttc agagtacaac
     6781 tattgatggt gaacgatcgc caatactagg ggcctttaaa acgggagcag ctattgcaac
     6841 cattgacgac tggtatcctg aagccactga gccactaagg gtcggacggt ttggggttca
     6901 tcgcgaagat gtcacttgct accgtcatcc gtctaccgga aaagattttt tctcgatatt
     6961 acaacaagca gagcactata ttgaagtgtt gagcgccaac aaaactcccg ctcaagaaac
     7021 tatcaacgac atgcactttt taatggctaa cctgattaag ggtgggatgt tccagcataa
     7081 aggagactga ctgtgaaatg gtattataag acaatcacct ttctgccaga gttgtgcaac
     7141 aacgagtcac tggctgcaaa gtgtctccgc gttctgcatg gatttaacta tcagtatgag
     7201 acacgaaata tcggcgtttc atttccgctt tggtgtgatg caacggttgg aaaaaagatt
     7261 tcatttgtca gcaagaacaa gatagaactc gacttactac ttaaacaaca ctatttcgtc
     7321 caaatggaac aacttcaata ctttcatata tccaacactg ttctcgtccc agaagattgt
     7381 acatacgttt cctttagacg ctgtcaatct atagataagc tcacagcagc agggctggca
     7441 aggaaaatca gacgcctgga gaaacgtgct ctatctagag gcgagcaatt tgacccatca
     7501 tcttttgctc aaaaagagca tactgcaata gcgcactacc actcacttgg ggagtccagc
     7561 aaacagacga accgcaactt tcgactcaat atcaggatgc tctcggagca accccgtgag
     7621 gggaactcga tttttagtag ctatggctta tcaaattcag aaaactcgtt tcagcctgta
     7681 cccttaatct aatagcattg gaagtggata acggatccgc gatcgcggcg cgccacctgg
     7741 tggccggccg gtaccacgcg tgcgcgctga tccggctgct aacaaagccc gaaaggaagc
     7801 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     7861 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg
     7921 tggtcgccat gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc
     7981 ggcggccaaa gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg
     8041 catatagcgc tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc
     8101 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg
     8161 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc
     8221 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag
     8281 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     8341 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     8401 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     8461 atgcttcaat aacattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     8521 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     8581 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     8641 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     8701 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     8761 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     8821 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     8881 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     8941 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     9001 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     9061 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     9121 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     9181 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     9241 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     9301 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     9361 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     9421 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     9481 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
