LOCUS       sequence-350785-        7556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MBP fused to Cas14a1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7556)
  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 7556)
  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: pLBH531_MBP-Cas14a1 expression
FEATURES             Location/Qualifiers
     source          1..7556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    93..232
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     148..167
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(247..264)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(418..1006)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(498..517)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1177..1968,1969..2037))
                     /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     1800..1819
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2038..2142)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2210..2228
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2255..2260,2261..2277,2278..2283)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(2649..2696)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2692..2710
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4432..4452)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(4507..5607)
                     /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     complement(4510..4533)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(5617..5646)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             5666..5688
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(5776..5795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5777..5795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5879..5898)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             7356..7547
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(join(7541..7556,1..7))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
       61 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
      121 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
      181 attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
      241 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
      301 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
      361 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      421 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      481 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      541 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      601 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      661 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      721 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      781 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      841 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      901 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
      961 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1021 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1081 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1141 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1201 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1261 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     1321 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1381 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1441 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat
     1501 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1561 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     1621 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     1681 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     1741 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga
     1801 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     1861 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     1921 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     1981 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2041 cttccttttt caatgttatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2101 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2161 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     2221 cacgaggccc tttcgtcttc aagaattctc atgtttgaca gcttatcatc gataagcttt
     2281 aatgcggtag tttatcacag ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta
     2341 acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc ataggcttgg
     2401 ttatgccggt actgccgggc ctcttgcggg atatcgtcca ttccgacagc atcgccagtc
     2461 actatggcgt gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg
     2521 gagcactgtc cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta cttggagcca
     2581 ctatcgacta cgcgatcatg gcgaccacac ccgtcctgtg gatatccgga tatagttcct
     2641 cctttcagca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctagttat
     2701 tgctcagcgg tggcagcagc caactcagct tcctttcggg ctttgttagc agccggatca
     2761 gcgcgcacgc gtggtaccgg ccggccacca ggtggcgcgc cgcgatcgcg gatccgttat
     2821 ccacttccaa tgttattacg gttcctcttt ggtgcttttc agtttcgggt ttgaaatatt
     2881 cagggctgca ttataatcgg cgttttcttt gaagttgcat ttttcgcatt taaagtgcgg
     2941 gaatttgttt ttcttgcggt actcgaagtt gaaatagttg ttcagatggc cacatttgct
     3001 acaggttttg ctggtattat tcggtgcaac tttacgaatt tcgatgccat actgcttcag
     3061 tttgaattcg attttgttct gcatttctgc atacggccaa aagccacgca ggcgaatgtt
     3121 aaaatagctg tcctctttac gtttcatgct ttccagattt tccatctgca cggtgccaac
     3181 tttgttttta atgaagaaat ccgcgatttc acatgcccaa cgttcaatca gctttttgcg
     3241 aaaacgttca cttttttcgg tcagaatggt gatcggtttc agtttgtttt ttgcaccatg
     3301 acctgcacgt ttatgacggt tttttttcag caggatacga cgacgtgcaa acattttctt
     3361 gttgaagtga aacaggtcgt tatcgctgat gctataacgg ctaaatgcat tgttaattgc
     3421 gcaaaccagc ggtgatttaa caccaacatc aataccacca ataatgctcg gatccacacc
     3481 tttatcaatt ttcggaacat caatgctcag attcagcatc catgcgcttt tttcacagat
     3541 tttgctgcca cgtttaactt caatgtagct ggtctgataa tcgccattca taactttttt
     3601 gatttcggct tcggtgcctt catctttgct ccaaccttta ttacgtttac gacgctgggt
     3661 gctcagcagc aggctaatcg gtttcgggct tttctgaacc tgttcaaagt caaatttctc
     3721 ccacggacga tatttatcaa tctctttttt gacctgccaa cgaccaaacg gaatcttgat
     3781 gatgaagtcg ctattatgat tgctaatttc aaaaccggta tactgaccac ctttctgttt
     3841 aaccagcgga atcggaaaat tatcgctttt ggtggtcggc agaccacttt tcatgttttt
     3901 cagttctttc agacgaaaat tgcttttgat tttgctacgc agaccgcttg caattgctgc
     3961 gtttttaaac agttctgctg cacgggtata acaaacatca ctcagataat gttcaacgct
     4021 gctggcattt gcaatacctt tgcctttgat aaaaatctcg tagtacagtt cgatcaggct
     4081 ctgattatag atttctgctg cctgtttctg cagctggcga aaaatttcgc tgatttcttg
     4141 ccaaaaaact gcatccggaa actgaccacg cagtttttgg tagaatttgt catccagttt
     4201 acgtgcttta caaaacaggc atgcattacg ttcaacctgt gtggtacaat atgctgcaac
     4261 tttcagatgt ttgctgcagg cttctttcac tttgtctttg ttcttttcca gtgcgatttt
     4321 ttcgcggttg tttttttcgt cggcaacaat tttttccact tctgcgctat tatacggacg
     4381 cacaatacgc agtttcagtg ttttggtaat ggtgtttttg gccattgcat tggattggaa
     4441 gtacaggttt tcctcgatcc cgaggttgtt gttattgtta ttgttgttgt tgttcgagct
     4501 cgaattagtc tgcgcgtctt tcagggcttc atcgacagtc tgacgaccgc tggcggcgtt
     4561 gatcaccgca gtacgcacgg cataccagaa agcggacatc tgcgggatgt tcggcatgat
     4621 ttcacctttc tgggcgtttt ccatagtggc ggcaatacgt ggatctttcg ccaactcttc
     4681 ctcgtaagac ttcagcgcta cggcacccag cggtttgtct ttattaaccg cttccagacc
     4741 ttcatcagtc agcagatagt tttcgaggaa ctcttttgcc agctctttgt tcggactggc
     4801 ggcgttaata cctgcgctca gcacgccaac gaacggtttg gatggttgac ccttgaaggt
     4861 cggcagtacc gttacaccat aattcacttt gctggtgtcg atgttggacc atgcccacgg
     4921 gccgttgatg gtcatcgctg tttcgccttt attaaaggca gcttctgcga tggagtaatc
     4981 ggtgtctgca ttcatgtgtt tgtttttaat caggtcaacc aggaaggtca gacccgcttt
     5041 cgcgccagcg ttatccacgc ccacgtcttt aatgtcgtac ttgccgtttt catacttgaa
     5101 cgcataaccc ccgtcagcag caatcagcgg ccaggtgaag tacggttctt gcaggttgaa
     5161 catcagcgcg ctcttacctt tcgctttcag ttctttatcc agcgccggga tctcttccca
     5221 ggtttttggc gggttcggca gcagatcttt gttataaatc agcgataacg cttcaacagc
     5281 gatcgggtaa gcaatcagct tgccgttgta acgtacggca tcccaggtaa acggatacag
     5341 cttgtcctgg aacgctttgt ccggggtgat ttcagccaac aggccagatt gagcgtagcc
     5401 accaaagcgg tcgtgtgccc agaagataat gtcagggcca tcgccagttg ccgcaacctg
     5461 tgggaatttc tcttccagtt tatccggatg ctcaacggtg actttaattc cggtatcttt
     5521 ctcgaatttc ttaccgactt cagcgagacc gttatagcct ttatcgccgt taatccagat
     5581 taccagttta ccttcttcga ttttcataga agaaccatgg tgatggtgat ggtgatggtg
     5641 atggtgagaa gatttcatat gtatatctcc ttcttaaagt taaacaaaat tatttctaga
     5701 actcgagtcc tgcagggcgg ccgcttaatt aaagagctct ccggagccgg cactagaggg
     5761 aaaccgttgt ggtctcccta tagtgagtcg tattaatttc gcgggatcga gatctcgatc
     5821 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     5881 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct
     5941 tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatctcc
     6001 ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc
     6061 ttcctaatgc aggagtcgca taagggagag cgtcgaccga tgcccttgag agccttcaac
     6121 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     6181 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     6241 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     6301 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     6361 gccattatcg ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg
     6421 cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat cgggatgccc
     6481 gcgttgcagg ccatgctgtc caggcaggta gatgacgacc atcagggaca gcttcaagga
     6541 tcgctcgcgg ctcttaccag cctaacttcg atcattggac cgctgatcgt cacggcgatt
     6601 tatgccgcct cggcgagcac atggaacggg ttggcatgga ttgtaggcgc cgccctatac
     6661 cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg
     6721 gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct
     6781 tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca
     6841 tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca
     6901 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     6961 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     7021 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     7081 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     7141 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     7201 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     7261 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     7321 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     7381 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     7441 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     7501 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcag
//
