LOCUS       sequence-181380-        9434 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 9434)
  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 9434)
  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-Lbu_C2c2_R1072A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     971..990
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1074..1093
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1074..1092
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1181..1203
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1223..1240
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1250..2350
                     /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     2324..2347
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2405..2425
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6037..6055)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6051..6098
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6464..6469,6470..6486,6487..6492))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6519..6537)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6605..6709
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6710..6778,6779..7570)
                     /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(6928..6947)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7741..8329
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8230..8249
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8483..8500
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8515..8654
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8580..8599)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8740..8762
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8756..8947)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt ctgccaaggg
       61 ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct tggagtggtg
      121 aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac
      181 cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc catgccaacc
      241 cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg
      301 aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta
      361 cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa
      421 tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg
      481 cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
      541 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
      601 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
      661 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
      721 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgacgct
      781 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
      841 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
      901 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
      961 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     1021 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
     1081 actcactata gggagaccac aacggtttcc ctctagtgcc ggctccggag agctctttaa
     1141 ttaagcggcc gccctgcagg actcgagttc tagaaataat tttgtttaac tttaagaagg
     1201 agatatacat atgaaatctt ctcaccatca ccatcaccat ggttcttcta tgaaaatcga
     1261 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
     1321 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
     1381 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
     1441 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
     1501 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
     1561 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc
     1621 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
     1681 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
     1741 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
     1801 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
     1861 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
     1921 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
     1981 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
     2041 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
     2101 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     2161 gctgaagtct tacgaggaag agttggcgaa agatccacgt attgccgcca ctatggaaaa
     2221 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     2281 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     2341 cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat
     2401 cgaggaaaac ctgtacttcc aatccaatgc aatgaaagtg acgaaggtag gaggcatttc
     2461 gcataagaag tacacgtccg aaggccgctt agtgaagtca gaatcggaag aaaatcgcac
     2521 agacgaacgt ctgtcggcgt tgcttaatat gcgccttgac atgtatatca agaatcccag
     2581 cagcacggaa accaaggaaa atcaaaaacg cattgggaaa ttaaagaaat tcttctcaaa
     2641 caaaatggtc tatcttaaag acaatacctt gagtttgaag aatgggaaaa aggagaacat
     2701 tgatcgtgag tattctgaga ctgacatcct tgagagcgat gtccgtgaca agaaaaactt
     2761 cgccgtgttg aaaaagatct atctgaatga aaacgtgaac tcggaggaat tggaagtttt
     2821 tcgtaacgac attaagaaga aactgaacaa aatcaacagc ctgaagtact catttgaaaa
     2881 gaataaggcg aattatcaaa agattaatga gaataacatc gagaaggttg aaggtaagtc
     2941 aaagcgtaac attatttacg attattatcg tgagtcagcg aaacgtgacg cttatgtaag
     3001 caatgtgaaa gaagcctttg ataagcttta caaggaagag gacattgcaa aacttgttct
     3061 tgaaattgag aaccttacga agttagagaa atacaagatt cgcgagttct accacgaaat
     3121 tattggacgt aagaatgaca aggaaaactt tgcaaaaatc atctacgaag aaatccagaa
     3181 tgttaataac atgaaagagt tgatcgagaa ggtaccggac atgagtgagt tgaaaaagag
     3241 ccaagtattt tacaagtatt acttagacaa agaagagttg aacgacaaga acatcaaata
     3301 cgcgttttgt catttcgtgg aaatcgaaat gagtcagttg ctgaagaact acgtatataa
     3361 gcgcttaagt aatatctcga atgacaaaat taagcgtatc tttgaatacc agaacttgaa
     3421 aaaattgatc gaaaataagc tgttaaacaa acttgacacg tacgtccgta attgtggaaa
     3481 gtataattat tatttgcaag acggcgaaat tgccacttca gatttcatcg cccgcaaccg
     3541 tcagaatgaa gcgtttcttc gcaacatcat tggggtgtca tctgtggcct acttttctct
     3601 tcgcaacatt cttgaaacgg agaacgagaa tgatattact gggcgtatgc gcggcaaaac
     3661 agttaagaac aataaaggtg aagagaagta cgtgtccgga gaagttgata agatctataa
     3721 tgaaaataag aagaacgagg ttaaggagaa cttaaaaatg ttctattcgt acgatttcaa
     3781 tatggacaac aagaatgaaa tcgaagattt cttcgccaac atcgacgagg cgatttcttc
     3841 catccgtcac ggtattgtcc acttcaactt ggaattagaa ggtaaggata tctttgcgtt
     3901 caagaacatt gcgccatccg aaatctcaaa gaagatgttt cagaatgaga ttaacgagaa
     3961 aaaactgaaa ttgaagatct ttcgtcaact gaactctgcc aacgtgttcc gctatctcga
     4021 aaagtataaa attctgaatt accttaaacg tacacgcttc gagtttgtca ataaaaatat
     4081 cccattcgtc ccgtctttca ccaaattata ttcgcgcatt gatgacctga agaatagtct
     4141 tgggatttac tggaaaactc cgaaaacaaa cgacgacaat aagactaagg agattattga
     4201 tgcccaaatc tatttgctta aaaacatcta ttacggggag ttcctgaatt atttcatgtc
     4261 gaacaatggt aatttctttg agatttctaa agaaatcatc gaattgaaca agaacgataa
     4321 acgcaactta aagactgggt tttacaagct gcaaaagttt gaagacatcc aggagaagat
     4381 tccaaaggaa tacttggcga atatccagtc cctgtacatg attaatgccg gtaatcagga
     4441 cgaagaagaa aaggacactt atattgattt cattcaaaag atcttcttaa agggatttat
     4501 gacgtatctt gctaataacg gtcgtttaag tctgatttac atcggctcgg atgaagaaac
     4561 aaatacgtca ttagcagaaa agaagcaaga gtttgacaag ttcttgaaga agtacgagca
     4621 gaacaataat atcaagatcc cctatgagat caatgaattc ctgcgtgaga tcaaactggg
     4681 aaacatcctg aagtatactg agcgtttaaa catgttctac cttatcttaa agcttttgaa
     4741 tcacaaggag ctgacaaatc tgaagggtag tcttgaaaaa tatcagtctg ccaataagga
     4801 agaagcgttc tctgaccaat tggagttaat taacctgctt aaccttgaca acaaccgcgt
     4861 gacggaagac ttcgaattag aggccgacga gattggaaaa tttcttgatt tcaatggcaa
     4921 caaagttaag gataacaagg aactgaaaaa gttcgataca aacaagatct actttgacgg
     4981 cgagaacatt atcaaacacc gtgccttcta caatattaag aaatatggca tgttaaactt
     5041 actggagaaa attgccgaca aggctggata caagatctcg atcgaagagc tgaagaaata
     5101 ctccaataaa aagaatgaga tcgagaagaa ccataagatg caggaaaatc tgcaccgcaa
     5161 atacgctcgt ccccgtaaag acgagaagtt tacagatgag gactatgaaa gttacaagca
     5221 agctattgag aatattgagg agtacaccca ccttaagaac aaggtagaat tcaatgagct
     5281 gaatttactg cagggcctgt tgctgcgcat tttacatcgt ttagtcggat atacctcaat
     5341 ttgggaacgc gatctgcgct tccgccttaa aggtgagttc ccagaaaacc aatacatcga
     5401 agagatcttc aactttgaaa ataagaagaa cgtgaagtac aaagggggtc agattgtaga
     5461 gaaatacatt aaattctaca aggaattaca tcaaaatgat gaagttaaga tcaacaagta
     5521 cagttccgcg aatatcaagg tgttgaagca agaaaagaag gacctttata ttcgcaatta
     5581 catcgcccac ttcaattata ttcctcacgc cgagatctca ctgctggaag tccttgaaaa
     5641 tttggcaaaa ttgctgtcct acgatcgcaa actgaaaaat gccgtaatga aatcagtagt
     5701 tgatatcctt aaggagtatg gttttgtagc cacattcaaa atcggggcgg acaagaagat
     5761 cggtattcag acactggaga gcgaaaaaat cgtgcatctt aagaatctta agaagaagaa
     5821 gttaatgact gaccgcaatt ccgaggaact ttgcaaattg gtgaagatta tgtttgaata
     5881 caaaatggaa gagaaaaagt ctgaaaacta ataacattgg aagtggataa cggatccgcg
     5941 atcgcggcgc gccacctggt ggccggccgg taccacgcgt gcgcgctgat ccggctgcta
     6001 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     6061 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     6121 gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc
     6181 gaagcgagca ggactgggcg gcggccaaag cggtcggaca gtgctccgag aacgggtgcg
     6241 catagaaatt gcatcaacgc atatagcgct agcagcacgc catagtgact ggcgatgctg
     6301 tcggaatgga cgatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct
     6361 acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac
     6421 ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg
     6481 ataagctgtc aaacatgaga attcttgaag atgaaagggc ctcgtgatac gcctattttt
     6541 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     6601 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     6661 gagacaataa ccctgataaa tgcttcaata acattgaaaa aggaagagta tgagtattca
     6721 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     6781 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     6841 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     6901 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     6961 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     7021 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     7081 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     7141 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     7201 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     7261 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     7321 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     7381 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     7441 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     7501 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     7561 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     7621 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     7681 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     7741 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     7801 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     7861 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     7921 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     7981 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     8041 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     8101 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     8161 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     8221 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     8281 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     8341 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     8401 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     8461 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     8521 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     8581 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     8641 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     8701 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     8761 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     8821 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     8881 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     8941 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     9001 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     9061 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     9121 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     9181 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     9241 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     9301 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     9361 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     9421 acccaacgct gccc
//
