LOCUS       sequence-215707-        7490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 10xHis-MBP fused to Cas14b1 under t7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7490)
  AUTHORS   Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, 
            Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA
  TITLE     Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.
  JOURNAL   Science. 2018 Oct 18. pii: science.aav4294. doi: 
            10.1126/science.aav4294.
  PUBMED    30337455
REFERENCE   2  (bases 1 to 7490)
  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"; journalName: "Science. 
            2018 Oct 18. pii: science.aav4294. doi: 10.1126/science.aav4294."
COMMENT     Sequence Label: pLBH532_MBP-Cas14b1 expression
FEATURES             Location/Qualifiers
     source          1..7490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     56..75
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        56..74
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             163..185
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             205..234
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             244..1344
                     /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     1318..1341
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1399..1419
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3075..3093)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3089..3136
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3502..3507,3508..3524,3525..3530))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3557..3575)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3643..3747
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3748..3816,3817..4608)
                     /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(3966..3985)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4779..5367
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5268..5287
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5521..5538
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5553..5692
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5618..5637)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5778..5800
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5794..5985)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7443..7462
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
       61 cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg agagctcttt
      121 aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta actttaagaa
      181 ggagatatac atatgaaatc ttctcaccat caccatcacc atcaccatca ccatggttct
      241 tctatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
      301 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
      361 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
      421 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
      481 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
      541 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
      601 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
      661 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
      721 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
      781 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
      841 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
      901 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
      961 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
     1021 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag
     1081 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1141 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     1201 gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     1261 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     1321 gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac
     1381 aacaacctcg ggatcgagga aaacctgtac ttccaatcca atgcaatgat tgcccagaaa
     1441 accatcaaga ttaaactgaa tccgaccaaa gagcagatca tcaaactgaa cagcatcatc
     1501 gaagagtaca tcaaagtgag caatttcacc gccaaaaaaa tcgccgaaat ccaagaaagc
     1561 tttaccgata gcggtctgac ccagggcacc tgtagcgaat gtggtaaaga aaaaacctac
     1621 cgcaaatacc atctgctgaa aaaagacaac aaactgttct gcatcacgtg ctacaaacgt
     1681 aaatatagcc agtttaccct gcagaaggtg gaatttcaga ataaaaccgg tctgcgtaat
     1741 gttgcaaaac tgccgaaaac ctattatacc aatgcaattc gttttgccag cgataccttt
     1801 agcggttttg atgaaatcat caagaaaaag cagaatcgcc tgaatagcat tcagaaccgt
     1861 ctgaactttt ggaaagaact gctgtataat ccgagcaacc gcaacgaaat caaaatcaaa
     1921 gttgtgaagt atgcccctaa aaccgataca cgtgaacatc cgcactatta tagcgaagca
     1981 gaaattaaag gtcgcatcaa acgtctggaa aagcagctga aaaagttcaa aatgcccaag
     2041 tatccggaat ttaccagcga aaccattagc ctgcagcgtg aactgtatag ctggaaaaat
     2101 ccggatgaac tgaaaattag cagcatcacc gataaaaacg agagcatgaa ctattacggc
     2161 aaagagtatc tgaaacgcta tatcgatctg atcaatagcc agacaccgca gattctgtta
     2221 gaaaaagaaa acaacagctt ttatctgtgc ttcccgatca ccaaaaacat cgaaatgccg
     2281 aaaatcgatg atacctttga accggttggt attgattggg gtattacccg taatattgcc
     2341 gttgttagca ttctggatag caaaaccaaa aaaccgaagt tcgtgaaatt ctatagcgca
     2401 ggttatattc tgggtaaacg caaacactat aaaagcctgc gtaaacactt tggccagaaa
     2461 aaacgtcagg acaaaattaa caaactgggc accaaagaag atcgcttcat tgatagcaac
     2521 attcacaaac tggcctttct gatcgtgaaa gaaattcgca atcatagcaa caaaccgatc
     2581 atcctgatgg aaaacattac cgataatcgt gaagaggccg aaaaaagcat gcgtcagaac
     2641 attctgctgc atagcgttaa aagccgtctg cagaactata ttgcatataa agcactgtgg
     2701 aacaacattc cgaccaatct ggttaaaccg gaacatacca gccagatttg taatcgttgt
     2761 ggtcatcagg atcgcgaaaa ccgtccgaaa ggtagcaaac tgtttaaatg cgtgaaatgc
     2821 aactatatga gcaacgccga ttttaacgcc agcattaata tcgcacgcaa attttacatc
     2881 ggcgaatatg agccgttcta caaagataac gagaagatga aaagcggtgt caacagcatt
     2941 agcatgtaat aacattggaa gtggataacg gatccgcgat cgcggcgcgc cacctggtgg
     3001 ccggccggta ccacgcgtgc gcgctgatcc ggctgctaac aaagcccgaa aggaagctga
     3061 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     3121 cttgaggggt tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg
     3181 tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc
     3241 ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat
     3301 atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca
     3361 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     3421 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     3481 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
     3541 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3601 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3661 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3721 cttcaataac attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3781 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3841 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3901 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3961 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc
     4021 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4081 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4141 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4201 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4261 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta
     4321 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4381 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4441 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4501 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4561 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4621 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4681 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4741 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4801 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4861 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4921 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4981 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5041 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5101 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5161 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5221 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5281 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5341 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5401 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5461 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5521 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5581 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5641 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5701 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5761 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5821 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5881 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5941 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     6001 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     6061 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     6121 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     6181 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     6241 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6301 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6361 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6421 caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc ccgagatgcg
     6481 ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag ggttggtttg
     6541 cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg tgaatccgtt
     6601 agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc
     6661 aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa cccgttccat
     6721 gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat cgaagttagg
     6781 ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg
     6841 gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag aatcataatg
     6901 gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag cgcgtcggcc
     6961 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     7021 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     7081 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     7141 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     7201 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgacg ctctccctta
     7261 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     7321 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     7381 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     7441 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
//
