LOCUS       sequence-255645-        7190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V. cholerae CAST TniQ from a T7 promoter, with N-terminal 
            His10-MBP-TEVsite-StrepII for purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7190)
  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 7190)
  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: 
            "10.1038/s41586-019-1323-z"; journalName: "Nature"; date: "2019-07";
            volume: "571"; issue: "7764"; pages: "219-225"
COMMENT     Sequence Label: pSL0376 (His-MBP-StrepII-TniQ)
FEATURES             Location/Qualifiers
     source          1..7190
                     /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"
     CDS             4138..4161
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(5487..5505)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5501..5548
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5914..5919,5920..5936,5937..5942))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5969..5987)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6055..6159
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6160..6228,6229..7020)
                     /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(6378..6397)
                     /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 caatgcatgg
     4141 agccacccgc agttcgaaaa aggtggcagc ggttctatgt ttttgcaaag acctaaacct
     4201 tacagcgatg aaagtttaga aagtttcttt atccgagtgg ctaacaaaaa tggctacggt
     4261 gatgtccatc gcttcctaga agccactaaa cgattccttc aagacattga ccataatggc
     4321 tatcaaacct ttccgactga tataactcgg ataaacccat actcagctaa aaacagttcc
     4381 agcgcacgaa ctgcgtcatt cctgaaactt gcacaattga catttaatga accgccagag
     4441 ctacttgggt tggcaattaa cagaacaaac atgaaatact cgccgtcaac tagcgcggtt
     4501 gttagaggtg cagaagtctt tcctcgcagt ttactacgga cgcactccat cccctgctgt
     4561 cctttgtgtc tgcgagaaaa tggctacgcc tcctaccttt ggcactttca ggggtacgaa
     4621 tactgccaca gccataacgt acctttaatt accacttgta gctgtggtaa ggagtttgac
     4681 taccgagtat ctgggttaaa gggcatttgc tgcaaatgca aggagcctat caccttaacc
     4741 agcagggaga acggtcatga ggcagcgtgt actgtttcaa actggcttgc tggccatgaa
     4801 tctaaacctc tgccaaatct tcctaaaagc taccgatggg gtttagttca ttggtggatg
     4861 ggtattaaag atagcgagtt cgatcacttt tcgttcgttc aatttttctc aaactggcca
     4921 aggtcattcc actcgataat cgaagatgaa gtagagttca accttgagca tgctgttgtc
     4981 agcacgtctg aattacgact aaaagatctt cttggtcgat tgtttttcgg ttcaattcgg
     5041 ttacctgagc ggaatcttca acacaatatc atccttggtg agcttctctg ctatttagaa
     5101 aatcgtttat ggcaagacaa gggattaatc gccaacctca aaatgaacgc gttagaggcg
     5161 actgtaatgt taaattgtag cctcgatcag attgcatcaa tggttgaaca acgcatcttg
     5221 aagccaaatc gaaaaagcaa gcccaacagc cctcttgatg ttaccgatta tctatttcat
     5281 ttcggcgata ttttctgtct ttggttagct gagttccaaa gcgatgagtt taaccgttcg
     5341 ttttatgtgt cgaggtggta atagcattgg aagtggataa cggatccgcg atcgcggcgc
     5401 gccacctggt ggccggccgg taccacgcgt gcgcgctgat ccggctgcta acaaagcccg
     5461 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     5521 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatatccaca
     5581 ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc gaagcgagca
     5641 ggactgggcg gcggccaaag cggtcggaca gtgctccgag aacgggtgcg catagaaatt
     5701 gcatcaacgc atatagcgct agcagcacgc catagtgact ggcgatgctg tcggaatgga
     5761 cgatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     5821 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     5881 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     5941 aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6001 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     6061 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     6121 ccctgataaa tgcttcaata acattgaaaa aggaagagta tgagtattca acatttccgt
     6181 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6241 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6301 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6361 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     6421 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6481 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6541 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6601 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6661 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     6721 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6781 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6841 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6901 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6961 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     7021 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     7081 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     7141 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
