LOCUS       sequence-215705-        7580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 10xHis-MBP fused to Cas14b2 under t7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7580)
  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 7580)
  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: pLBH533_MBP-Cas14b2 expression
FEATURES             Location/Qualifiers
     source          1..7580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..98
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        79..97
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             186..208
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             228..257
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             267..1367
                     /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     1341..1364
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1422..1442
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(3188..3206)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3202..3249
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3615..3620,3621..3637,3638..3643))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3670..3688)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3756..3860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3861..3929,3930..4721)
                     /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(4079..4098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4892..5480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5381..5400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5634..5651
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5666..5805
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5731..5750)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5891..5913
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5907..6098)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     7556..7575
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
       61 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
      121 gtgccggctc cggagagctc tttaattaag cggccgccct gcaggactcg agttctagaa
      181 ataattttgt ttaactttaa gaaggagata tacatatgaa atcttctcac catcaccatc
      241 accatcacca tcaccatggt tcttctatga aaatcgaaga aggtaaactg gtaatctgga
      301 ttaacggcga taaaggctat aacggtctcg ctgaagtcgg taagaaattc gagaaagata
      361 ccggaattaa agtcaccgtt gagcatccgg ataaactgga agagaaattc ccacaggttg
      421 cggcaactgg cgatggccct gacattatct tctgggcaca cgaccgcttt ggtggctacg
      481 ctcaatctgg cctgttggct gaaatcaccc cggacaaagc gttccaggac aagctgtatc
      541 cgtttacctg ggatgccgta cgttacaacg gcaagctgat tgcttacccg atcgctgttg
      601 aagcgttatc gctgatttat aacaaagatc tgctgccgaa cccgccaaaa acctgggaag
      661 agatcccggc gctggataaa gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc
      721 tgcaagaacc gtacttcacc tggccgctga ttgctgctga cgggggttat gcgttcaagt
      781 atgaaaacgg caagtacgac attaaagacg tgggcgtgga taacgctggc gcgaaagcgg
      841 gtctgacctt cctggttgac ctgattaaaa acaaacacat gaatgcagac accgattact
      901 ccatcgcaga agctgccttt aataaaggcg aaacagcgat gaccatcaac ggcccgtggg
      961 catggtccaa catcgacacc agcaaagtga attatggtgt aacggtactg ccgaccttca
     1021 agggtcaacc atccaaaccg ttcgttggcg tgctgagcgc aggtattaac gccgccagtc
     1081 cgaacaaaga gctggcaaaa gagttcctcg aaaactatct gctgactgat gaaggtctgg
     1141 aagcggttaa taaagacaaa ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt
     1201 tggcgaaaga tccacgtatt gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc
     1261 cgaacatccc gcagatgtcc gctttctggt atgccgtgcg tactgcggtg atcaacgccg
     1321 ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc gcagactaat tcgagctcga
     1381 acaacaacaa caataacaat aacaacaacc tcgggatcga ggaaaacctg tacttccaat
     1441 ccaatgcaat ggaagaaagc attattaccg gtgtgaaatt caaactgcgc atcgataaag
     1501 aaaccaccaa aaaactgaac gagtacttcg atgaatatgg caaagcaatt aacttcgccg
     1561 tgaagatcat tcagaaagaa ctggcagatg atcgttttgc aggtaaagca aaactggacc
     1621 agaataaaaa cccgatcctg gatgaaaacg gcaaaaaaat ctatgaattc ccggatgaat
     1681 tttgcagctg tggtaaacag gttaacaagt acgttaacaa caaaccgttt tgccaagagt
     1741 gctataaaat ccgctttacc gaaaatggta ttcgcaaacg tatgtatagc gccaaaggtc
     1801 gtaaagccga acataaaatc aatatcctga acagcaccaa caagatcagc aaaacccatt
     1861 ttaactatgc cattcgcgaa gccttcattc tggataaaag catcaaaaag cagcgcaaaa
     1921 aacgtaatga acgtctgcgt gaaagtaaaa aacgtctgca gcagtttatc gatatgcgtg
     1981 atggtaaacg tgaaatttgc ccgaccatta aaggtcagaa agtggatcgt tttattcatc
     2041 cgagctggat caccaaagat aaaaagctgg aagattttcg cggttatacc ctgagcatta
     2101 tcaacagcaa aattaagatt ctggatcgca acatcaaacg cgaagaaaaa agcctgaaag
     2161 aaaaaggcca gatcatcttt aaagccaaac gtctgatgct ggataaatcc attcgttttg
     2221 ttggtgatcg caaagtgctg tttacaatta gtaaaaccct gccgaaagag tatgaactgg
     2281 atctgccgag caaagaaaaa cggctgaatt ggctgaaaga gaagatcgag attatcaaga
     2341 accagaaacc gaaatatgcc tatctgctgc gcaaaaacat tgagagcgaa aaaaaaccga
     2401 actatgagta ctatctgcag tacaccctgg aaattaaacc ggaactgaaa gatttttatg
     2461 atggtgccat tggtattgac cgtggcatta atcatattgc cgtttgcacc tttattagca
     2521 acgatggtaa agttacccct ccgaaatttt tcagcagcgg tgaaattctg cgtctgaaaa
     2581 atctgcagaa agagcgtgat cgctttctgc tgcgtaaaca caacaaaaat cgcaaaaaag
     2641 gcaacatgcg cgtgatcgaa aacaaaatca atctgatcct gcaccgttat agcaagcaga
     2701 ttgttgatat ggccaaaaag ctgaatgcca gcattgtttt tgaagaactg ggtcgtattg
     2761 gtaaaagccg caccaaaatg aaaaaaagcc agcgttataa actgagcctg ttcatcttca
     2821 agaaactgag cgatctggtt gattacaaaa gccgtcgtga aggtattcgt gttacctatg
     2881 ttccgcctga atataccagc aaagaatgta gccattgcgg tgaaaaagtt aatacccagc
     2941 gtccgtttaa tggcaactat agcctgttta aatgcaacaa atgtggcatc cagctgaaca
     3001 gcgattataa tgcaagcatc aacattgcga aaaagggcct gaaaattccg aatagcacct
     3061 aataacattg gaagtggata acggatccgc gatcgcggcg cgccacctgg tggccggccg
     3121 gtaccacgcg tgcgcgctga tccggctgct aacaaagccc gaaaggaagc tgagttggct
     3181 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     3241 ggttttttgc tgaaaggagg aactatatcc ggatatccac aggacgggtg tggtcgccat
     3301 gatcgcgtag tcgatagtgg ctccaagtag cgaagcgagc aggactgggc ggcggccaaa
     3361 gcggtcggac agtgctccga gaacgggtgc gcatagaaat tgcatcaacg catatagcgc
     3421 tagcagcacg ccatagtgac tggcgatgct gtcggaatgg acgatatccc gcaagaggcc
     3481 cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat
     3541 gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg
     3601 tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aattcttgaa
     3661 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3721 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3781 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3841 aacattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3901 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3961 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     4021 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     4081 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     4141 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     4201 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     4261 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4321 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4381 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     4441 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4501 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4561 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4621 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4681 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4741 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4801 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4861 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4921 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4981 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     5041 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5101 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5161 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5221 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     5281 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5341 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5401 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5461 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5521 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5581 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5641 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5701 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5761 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     5821 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5881 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5941 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     6001 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     6061 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     6121 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     6181 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     6241 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     6301 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     6361 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     6421 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     6481 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     6541 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     6601 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     6661 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     6721 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     6781 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     6841 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     6901 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     6961 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     7021 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     7081 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     7141 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     7201 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     7261 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     7321 aggagctgac tgggttgaag gctctcaagg gcatcggtcg acgctctccc ttatgcgact
     7381 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     7441 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     7501 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     7561 tgtcggcgat ataggcgcca
//
