LOCUS       sequence-181382-        9504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for Cas13a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9504)
  AUTHORS   East-Seletsky A, O'Connell MR, Burstein D, Knott GJ, Doudna JA
  TITLE     RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas 
            Enzymes.
  JOURNAL   Mol Cell. 2017 May 4;66(3):373-383.e3. doi: 
            10.1016/j.molcel.2017.04.008.
  PUBMED    28475872
REFERENCE   2  (bases 1 to 9504)
  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: 
            "10.1016/j.molcel.2017.04"; journalName: "Mol Cell"; date: 
            "2017-05-4- 4"; volume: "66"; issue: "3"; pages: "373-383.e3"
COMMENT     Sequence Label: p2CT-His-MBP-Lwa_Cas13a_WT
FEATURES             Location/Qualifiers
     source          1..9504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1039..1058
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1142..1161
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1142..1160
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1249..1271
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1291..1308
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1318..2418
                     /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     2392..2415
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2473..2493
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6175..6193)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6189..6236
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6602..6607,6608..6624,6625..6630))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6657..6675)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6743..6847
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6848..6916,6917..7708)
                     /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(7066..7085)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7879..8467
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8368..8387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8621..8638
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8653..8792
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8718..8737)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8878..8900
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8894..9085)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
       61 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
      121 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
      181 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
      241 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
      301 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
      361 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
      421 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
      481 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
      541 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
      601 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
      661 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
      721 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
      781 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
      841 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
      901 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
      961 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     1021 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     1081 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     1141 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
     1201 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
     1261 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatgg ttcttctatg
     1321 aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc
     1381 gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg
     1441 gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc
     1501 ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc
     1561 ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac
     1621 ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat
     1681 ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa
     1741 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg
     1801 attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac
     1861 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa
     1921 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc
     1981 gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg
     2041 aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc
     2101 gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc
     2161 gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt
     2221 gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat tgccgccact
     2281 atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg
     2341 tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc
     2401 ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca acaataacaa taacaacaac
     2461 ctcgggatcg aggaaaacct gtacttccaa tccaatgcaa tgtatatgaa gattacaaag
     2521 attgatggag tatcacatta taaaaaacaa gacaagggca tcttgaaaaa gaagtggaag
     2581 gacctggacg aacgcaagca gcgcgagaaa atcgaggcgc gctataacaa acagatcgag
     2641 tcgaaaattt acaaggaatt ctttcgcctg aagaacaaga agcgtatcga gaaagaggaa
     2701 gatcagaata ttaaaagctt atactttttc attaaggagt tgtacttgaa tgaaaagaat
     2761 gaagaatggg agctgaagaa tattaatctg gagatcttag atgacaagga acgtgttatt
     2821 aaggggtaca aattcaaaga ggatgtgtat ttttttaaag aaggctataa ggagtattat
     2881 ctgcgcattc tgttcaacaa cttgattgag aaagtacaaa atgagaatcg tgaaaaggta
     2941 cgcaagaaca aagagttctt ggatctgaaa gagattttca agaaatacaa aaaccgcaag
     3001 attgatctgt tactgaagtc aatcaacaat aacaagatta acctggagta taaaaaagaa
     3061 aatgtcaatg aggagatcta tggaattaac ccgaccaacg accgcgaaat gactttttat
     3121 gaattgctga aggagattat tgaaaagaag gatgaacaga aatcgatctt ggaggagaaa
     3181 ttagacaatt ttgatattac gaacttcctt gaaaatatcg aaaaaatctt caatgaggaa
     3241 acggagatca atatcatcaa aggtaaggta ttgaatgagc ttcgtgaata tatcaaagaa
     3301 aaggaagaaa acaactccga caacaagtta aagcagatct acaacttgga gcttaaaaag
     3361 tatatcgaga acaacttctc ctacaaaaag caaaagagta aatcgaagaa cggcaagaat
     3421 gactacctgt acctgaactt cctcaagaag attatgttca tcgaagaagt ggatgagaag
     3481 aaagagatca acaaggaaaa attcaaaaac aagatcaaca gtaactttaa gaacctgttt
     3541 gtccagcaca ttttggacta tgggaaactg ctgtattata aggagaacga cgaatacatc
     3601 aagaacacag ggcaacttga aacaaaggat ttggagtata ttaaaactaa ggaaactctt
     3661 atccgcaaga tggcggtgtt agtctctttt gcagcgaaca gctattacaa cctgtttggg
     3721 cgcgtttctg gggacatttt ggggaccgaa gtagtaaaat cttctaagac gaacgtcatc
     3781 aaggttggat cacacatttt caaggaaaag atgttgaatt acttctttga tttcgaaatc
     3841 ttcgatgcta acaagatcgt cgagatcttg gaatctattt catactccat ctataatgta
     3901 cgcaatggag tcgggcattt caataagtta attttgggca aatataagaa gaaagacatt
     3961 aatactaata agcgcatcga agaagacctg aacaacaatg aagaaatcaa gggttacttc
     4021 atcaagaaac gtggcgagat cgaacgcaaa gttaaggaga agtttttgtc caataacctg
     4081 caatattact atagcaaaga aaagattgag aattatttcg aggtgtacga attcgagatt
     4141 ctgaaacgca agatcccctt tgctccgaac ttcaaacgta ttatcaaaaa aggggaggac
     4201 ttgttcaata acaaaaacaa caagaagtac gaatacttca agaatttcga taagaattcg
     4261 gcggaagaaa agaaggagtt tctgaaaact cgcaattttt tacttaaaga gctttactac
     4321 aataactttt acaaagagtt cctgagtaag aaagaagaat tcgagaagat tgtattggaa
     4381 gtaaaggagg agaaaaagtc tcgcggcaat attaacaaca agaaatccgg ggtatctttc
     4441 cagagcatcg atgattatga cactaagatt aacatcagcg attatatcgc atcgattcac
     4501 aaaaaagaaa tggagcgtgt agagaagtac aatgaagaaa agcagaagga taccgcaaag
     4561 tacattcgtg actttgtcga agagatcttc ctgactggtt tcatcaatta cttggagaaa
     4621 gataaacgcc tgcacttctt aaaagaagaa ttctctatct tgtgtaataa taacaacaac
     4681 gtcgtggact tcaacatcaa cattaacgaa gagaagatta aagagttcct taaagaaaat
     4741 gatagcaaga cgctgaacct ttacttattc ttcaatatga ttgactcgaa gcgtatctct
     4801 gagtttcgca atgagcttgt taagtacaag caatttacga agaagcgttt agatgaggag
     4861 aaagaattct taggtattaa gatcgaatta tacgagacct taattgaatt cgtgatcctg
     4921 acccgcgaga aactggacac gaaaaaatcc gaggaaattg acgcatggct ggtagataag
     4981 ctgtatgtga aggactctaa tgagtataag gagtatgagg aaatccttaa attgtttgta
     5041 gacgaaaaaa ttttgtcctc taaagaggct ccctattacg ctaccgacaa caaaactcct
     5101 attttactga gtaacttcga gaaaacacgt aaatacggaa cgcaatcctt cttgtccgag
     5161 atccagagca attacaagta ttccaaagtg gagaaggaga atatcgaaga ttacaacaag
     5221 aaagaggaaa tcgagcagaa gaaaaagtcg aatatcgaaa agttacaaga tttaaaggtc
     5281 gaacttcaca agaaatggga gcaaaataag atcaccgaaa aggagattga aaagtacaat
     5341 aataccacgc gtaaaatcaa tgaatacaac tacttgaaga ataaggaaga gttgcagaac
     5401 gtatatttgt tacatgagat gttaagcgat ttacttgcac gcaatgtggc tttcttcaac
     5461 aagtgggagc gtgattttaa gttcattgtt attgctatca aacaattctt acgtgagaat
     5521 gacaaagaaa aagtcaacga atttcttaat cctccggaca actccaaggg caaaaaagtt
     5581 tacttttcag taagcaagta taaaaacaca gtcgaaaaca ttgacggaat tcacaagaac
     5641 ttcatgaacc tgatctttct gaacaacaag ttcatgaacc gcaagattga caaaatgaac
     5701 tgtgcaatct gggtttattt ccgtaattat attgcacatt ttttacacct gcatacaaag
     5761 aacgaaaaaa tctccttaat cagccagatg aatcttctga tcaaattatt tagctacgac
     5821 aaaaaagtcc aaaaccatat tttaaagtct accaagacgt tattggagaa atacaacatc
     5881 cagatcaact tcgagatctc caacgacaaa aatgaagttt tcaagtataa gatcaaaaat
     5941 cgcttgtata gtaagaaggg caagatgttg gggaagaaca ataaattcga gatcttagag
     6001 aatgagttct tggagaacgt gaaggcaatg ctggaatata gtgaataata acattggaag
     6061 tggataacgg atcccgcgat cgcggcgcgc cacctggtgg ccggccggta ccacgcgtgc
     6121 gcgctgatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     6181 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     6241 aaggaggaac tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg
     6301 atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt
     6361 gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca
     6421 tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc
     6481 ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca
     6541 ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg
     6601 cattaaagct tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct
     6661 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     6721 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6781 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag
     6841 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6901 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6961 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     7021 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     7081 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     7141 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     7201 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     7261 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     7321 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     7381 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     7441 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7501 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7561 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     7621 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7681 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7741 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7801 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7861 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7921 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     7981 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     8041 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     8101 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     8161 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     8221 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     8281 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8341 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8401 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8461 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     8521 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     8581 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     8641 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     8701 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     8761 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     8821 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     8881 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     8941 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     9001 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     9061 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     9121 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     9181 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     9241 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     9301 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     9361 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     9421 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     9481 gctaaccagt aaggcaaccc cgcc
//
