LOCUS       sequence-181510-        9419 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 9419)
  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 9419)
  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-Ppr_Cas13a_WT
FEATURES             Location/Qualifiers
     source          1..9419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     117..134
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    149..288
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(214..233)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     374..396
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(390..581)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2039..2058
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2142..2161
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2142..2160
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2249..2271
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2291..2308
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2318..3418
                     /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     3392..3415
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3473..3493
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7090..7108)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7104..7151
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7517..7522,7523..7539,7540..7545))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7572..7590)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7658..7762
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7763..7831,7832..8623)
                     /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(7981..8000)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8794..9382
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9283..9302
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
       61 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
      121 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
      181 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      241 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
      301 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
      361 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
      421 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
      481 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
      541 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
      601 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
      661 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
      721 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
      781 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
      841 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
      901 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
      961 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     1021 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     1081 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     1141 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     1201 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     1261 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     1321 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     1381 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     1441 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     1501 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     1561 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     1621 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     1681 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     1741 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     1801 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct cccttatgcg
     1861 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     1921 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     1981 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     2041 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     2101 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     2161 gagaccacaa cggtttccct ctagtgccgg ctccggagag ctctttaatt aagcggccgc
     2221 cctgcaggac tcgagttcta gaaataattt tgtttaactt taagaaggag atatacatat
     2281 gaaatcttct caccatcacc atcaccatgg ttcttctatg aaaatcgaag aaggtaaact
     2341 ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt
     2401 cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt
     2461 cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt
     2521 tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga
     2581 caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc
     2641 gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa
     2701 aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct
     2761 gatgttcaac ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta
     2821 tgcgttcaag tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg
     2881 cgcgaaagcg ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga
     2941 caccgattac tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa
     3001 cggcccgtgg gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact
     3061 gccgaccttc aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa
     3121 cgccgccagt ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga
     3181 tgaaggtctg gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta
     3241 cgaggaagag ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg
     3301 tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt
     3361 gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa
     3421 ttcgagctcg aacaacaaca acaataacaa taacaacaac ctcgggatcg aggaaaacct
     3481 gtacttccaa tccaatgcaa tgcgtgtgtc gaaagtgaaa gttaaagatg ggggcaaaga
     3541 caagatggtc ttggtccatc gtaagacgac cggtgcccaa cttgtttatt cagggcagcc
     3601 cgttagcaac gagacgtcta atattctgcc agaaaaaaag cgccagtcat tcgaccttag
     3661 caccctgaac aaaacaatta tcaaattcga tactgcgaaa aagcaaaagt taaatgtgga
     3721 ccagtataag atcgtagaaa agatcttcaa gtatcccaag caggagctgc ccaagcaaat
     3781 taaagcagag gagatcctgc cgtttttgaa tcacaaattc caggagccag tcaagtattg
     3841 gaaaaacggg aaggaagaga gcttcaattt aacattattg atcgtcgagg cagtccaagc
     3901 acaggacaag cgtaaacttc agccctatta tgactggaag acctggtaca tccaaactaa
     3961 aagcgattta ctgaagaaga gtatcgagaa caatcgtatc gatttgactg agaacttgtc
     4021 aaagcgcaag aaggcattgt tggcctggga gacagagttc acagcatccg gttcaatcga
     4081 cctgacgcat taccataagg tctatatgac ggatgttctg tgcaaaatgt tacaagatgt
     4141 taagccgctg acagatgata aagggaagat caacaccaat gcgtatcatc gtgggcttaa
     4201 gaaagccctg cagaaccacc aaccagccat ctttggaaca cgcgaggtgc ctaatgaggc
     4261 taatcgtgca gacaatcagt tgagcattta tcatttggag gtcgtgaagt atcttgagca
     4321 ctattttcca attaaaactt cgaaacgccg caataccgcc gacgatatcg ctcattattt
     4381 aaaggcacag acgttgaaga caacgattga aaagcagctg gtaaacgcca tccgcgcgaa
     4441 catcatccaa caaggcaaaa ccaatcatca cgagttgaaa gcggacacaa catcgaatga
     4501 tttgatccgt attaaaacta acgaagcctt cgtcttaaac ttaactggta cctgtgcgtt
     4561 tgcagcgaac aatattcgca acatggtgga caacgagcaa actaacgaca tcctgggaaa
     4621 gggggacttt atcaagagct tactgaagga caatacgaac agccaattgt actcgttttt
     4681 ttttggtgaa ggattatcca cgaataaggc agagaaagag acgcaacttt ggggtatccg
     4741 tggggctgtt caacagatcc gcaataatgt aaatcattac aagaaagacg cccttaaaac
     4801 cgtgttcaac atctcaaatt tcgagaatcc caccattact gacccgaaac aacagactaa
     4861 ctatgcagac accatctata aagcgcgctt catcaatgaa ttggaaaaga tccctgaagc
     4921 ctttgcacag cagcttaaaa ctggcggtgc tgtctcctat tacactatcg aaaacctgaa
     4981 atcattattg actacgtttc agttttcgct gtgtcgtagc acgattccat ttgcacctgg
     5041 tttcaaaaag gtttttaatg gaggaatcaa ttaccaaaac gccaagcaag atgaatcttt
     5101 ctatgagctt atgctggaac aatacttgcg taaagagaat ttcgccgaag agagctacaa
     5161 tgcacgttat tttatgctga agttgatcta taacaatctt ttcctgccgg gctttacgac
     5221 cgaccgtaag gcgttcgccg acagtgtggg atttgtgcaa atgcagaaca aaaagcaagc
     5281 tgagaaagtc aaccctcgca agaaagaggc gtatgcgttc gaggcggtac gtccgatgac
     5341 cgcagcagac tctatcgcag attacatggc atacgttcaa agtgaattaa tgcaggagca
     5401 aaacaaaaag gaggaaaaag ttgccgagga aactcgcatc aattttgaaa agttcgtcct
     5461 tcaggtgttc attaaagggt tcgattcgtt cttgcgcgcc aaagagtttg acttcgtcca
     5521 aatgccacaa cctcaattga ctgctacggc gtccaaccaa caaaaagccg acaagttaaa
     5581 ccaactggaa gcctctatta ctgctgattg caaacttacg ccccaatatg cgaaagcgga
     5641 cgatgctaca catatcgcgt tctacgtctt ttgcaaatta ctggatgcgg cacacttgtc
     5701 taatttgcgt aacgagctga ttaagtttcg cgaatccgtt aacgaattca aattccacca
     5761 tttgctggaa atcattgaga tttgcctttt atctgcagat gttgtgccaa cagactatcg
     5821 tgatctttat agtagcgaag ctgactgctt agcacgtctg cgcccgttca ttgaacaggg
     5881 cgcagacatc acaaactggt cagacttatt tgtacaatct gacaaacaca gcccagtcat
     5941 ccacgcaaac attgagctta gtgttaaata tggcacaact aaactgttag agcagattat
     6001 caataaagat actcaattca agaccacaga ggcaaatttt accgcctgga atactgcgca
     6061 gaagagcatc gaacagctta ttaagcaacg cgaggaccat cacgagcaat gggtcaaggc
     6121 taaaaacgcc gacgacaaag aaaaacaaga gcgcaaacgc gagaagtcca actttgcgca
     6181 aaagtttatt gagaaacacg gtgacgatta tcttgatatc tgcgattata tcaacactta
     6241 taactggctt gacaataaaa tgcacttcgt gcacctgaac cgtctgcacg gcttgactat
     6301 cgagctttta ggtcgcatgg ccgggtttgt cgccctgttt gatcgcgatt ttcaattttt
     6361 cgatgagcaa cagatcgctg acgagttcaa gttacacgga tttgtgaact tacacagtat
     6421 tgacaagaag ctcaatgaag tccctactaa aaagatcaaa gagatctacg acattcgtaa
     6481 caaaatcatt caaattaacg gaaacaagat caatgagtcc gtgcgtgcca acttaattca
     6541 atttatctcg tctaaacgta attactataa taacgccttc ttgcacgtgt ccaatgatga
     6601 gatcaaagaa aagcagatgt atgatatccg caaccatatc gcgcacttca actatttgac
     6661 gaaggacgcg gctgacttct ctttgattga cttgattaac gagcttcgcg aattgttaca
     6721 ttatgatcgc aaacttaaga acgctgtttc caaggccttt atcgacctgt ttgataagca
     6781 cggcatgatt ctgaagctta agcttaacgc agaccacaag ttaaaggttg agtccctgga
     6841 gccaaagaaa atttaccatc tgggatcatc tgcgaaggac aagccggagt atcaatattg
     6901 caccaaccaa gtcatgatgg catactgtaa catgtgccgc tcccttttag agatgaaaaa
     6961 ataataacat tggaagtgga taacggatcc gcgatcgcgg cgcgccacct ggtggccggc
     7021 cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     7081 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     7141 ggggtttttt gctgaaagga ggaactatat ccggatatcc acaggacggg tgtggtcgcc
     7201 atgatcgcgt agtcgatagt ggctccaagt agcgaagcga gcaggactgg gcggcggcca
     7261 aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa attgcatcaa cgcatatagc
     7321 gctagcagca cgccatagtg actggcgatg ctgtcggaat ggacgatatc ccgcaagagg
     7381 cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg
     7441 atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac
     7501 tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg
     7561 aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     7621 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     7681 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     7741 ataacattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     7801 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     7861 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     7921 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     7981 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     8041 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     8101 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     8161 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     8221 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     8281 cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac
     8341 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     8401 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     8461 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     8521 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     8581 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     8641 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     8701 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     8761 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     8821 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     8881 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     8941 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     9001 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     9061 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     9121 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     9181 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     9241 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     9301 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     9361 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcct
//
