LOCUS       sequence-255646-        7151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V. cholerae CAST TniQ from a T7 promoter, with N-terminal 
            His10-MBP-TEVsite tag for purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7151)
  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 7151)
  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: pSL0373 (His-MBP-TniQ)
FEATURES             Location/Qualifiers
     source          1..7151
                     /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(5448..5466)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5462..5509
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5875..5880,5881..5897,5898..5903))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5930..5948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6016..6120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6121..6189,6190..6981)
                     /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(6339..6358)
                     /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 tttttgcaaa gacctaaacc ttacagcgat gaaagtttag aaagtttctt tatccgagtg
     4201 gctaacaaaa atggctacgg tgatgtccat cgcttcctag aagccactaa acgattcctt
     4261 caagacattg accataatgg ctatcaaacc tttccgactg atataactcg gataaaccca
     4321 tactcagcta aaaacagttc cagcgcacga actgcgtcat tcctgaaact tgcacaattg
     4381 acatttaatg aaccgccaga gctacttggg ttggcaatta acagaacaaa catgaaatac
     4441 tcgccgtcaa ctagcgcggt tgttagaggt gcagaagtct ttcctcgcag tttactacgg
     4501 acgcactcca tcccctgctg tcctttgtgt ctgcgagaaa atggctacgc ctcctacctt
     4561 tggcactttc aggggtacga atactgccac agccataacg tacctttaat taccacttgt
     4621 agctgtggta aggagtttga ctaccgagta tctgggttaa agggcatttg ctgcaaatgc
     4681 aaggagccta tcaccttaac cagcagggag aacggtcatg aggcagcgtg tactgtttca
     4741 aactggcttg ctggccatga atctaaacct ctgccaaatc ttcctaaaag ctaccgatgg
     4801 ggtttagttc attggtggat gggtattaaa gatagcgagt tcgatcactt ttcgttcgtt
     4861 caatttttct caaactggcc aaggtcattc cactcgataa tcgaagatga agtagagttc
     4921 aaccttgagc atgctgttgt cagcacgtct gaattacgac taaaagatct tcttggtcga
     4981 ttgtttttcg gttcaattcg gttacctgag cggaatcttc aacacaatat catccttggt
     5041 gagcttctct gctatttaga aaatcgttta tggcaagaca agggattaat cgccaacctc
     5101 aaaatgaacg cgttagaggc gactgtaatg ttaaattgta gcctcgatca gattgcatca
     5161 atggttgaac aacgcatctt gaagccaaat cgaaaaagca agcccaacag ccctcttgat
     5221 gttaccgatt atctatttca tttcggcgat attttctgtc tttggttagc tgagttccaa
     5281 agcgatgagt ttaaccgttc gttttatgtg tcgaggtggt aatagcattg gaagtggata
     5341 acggatccgc gatcgcggcg cgccacctgg tggccggccg gtaccacgcg tgcgcgctga
     5401 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     5461 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     5521 aactatatcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg
     5581 ctccaagtag cgaagcgagc aggactgggc ggcggccaaa gcggtcggac agtgctccga
     5641 gaacgggtgc gcatagaaat tgcatcaacg catatagcgc tagcagcacg ccatagtgac
     5701 tggcgatgct gtcggaatgg acgatatccc gcaagaggcc cggcagtacc ggcataacca
     5761 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag
     5821 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa
     5881 gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg cctcgtgata
     5941 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     6001 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     6061 tatccgctca tgagacaata accctgataa atgcttcaat aacattgaaa aaggaagagt
     6121 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     6181 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6241 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     6301 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6361 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6421 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6481 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6541 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6601 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6661 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6721 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6781 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6841 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6901 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6961 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     7021 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     7081 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     7141 aaaggatctt c
//
