LOCUS       sequence-181376-        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_K1080A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      147..735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     636..655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     889..906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    921..1060
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(986..1005)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1146..1168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1162..1353)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2811..2830
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2914..2933
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2914..2932
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3021..3043
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3063..3080
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3090..4190
                     /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     4164..4187
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4245..4265
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7877..7895)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7891..7938
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8304..8309,8310..8326,8327..8332))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8359..8377)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8445..8549
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8550..8618,8619..9410)
                     /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(8768..8787)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
       61 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
      121 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
      181 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      241 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
      301 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
      361 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      421 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      481 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      541 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      601 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      661 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      721 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      781 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      841 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      901 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
      961 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     1021 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     1081 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     1141 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     1201 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     1261 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     1321 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     1381 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     1441 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     1501 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     1561 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     1621 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     1681 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     1741 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     1801 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     1861 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     1921 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     1981 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
     2041 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     2101 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     2161 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     2221 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     2281 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     2341 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     2401 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     2461 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     2521 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     2581 ctgactgggt tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc
     2641 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     2701 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     2761 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     2821 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     2881 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
     2941 aacggtttcc ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg
     3001 actcgagttc tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt
     3061 ctcaccatca ccatcaccat ggttcttcta tgaaaatcga agaaggtaaa ctggtaatct
     3121 ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag
     3181 ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
     3241 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
     3301 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt
     3361 atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
     3421 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg
     3481 aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca
     3541 acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
     3601 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
     3661 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt
     3721 actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
     3781 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct
     3841 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca
     3901 gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
     3961 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
     4021 agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca
     4081 tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg
     4141 ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct
     4201 cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac ctgtacttcc
     4261 aatccaatgc aatgaaagtg acgaaggtag gaggcatttc gcataagaag tacacgtccg
     4321 aaggccgctt agtgaagtca gaatcggaag aaaatcgcac agacgaacgt ctgtcggcgt
     4381 tgcttaatat gcgccttgac atgtatatca agaatcccag cagcacggaa accaaggaaa
     4441 atcaaaaacg cattgggaaa ttaaagaaat tcttctcaaa caaaatggtc tatcttaaag
     4501 acaatacctt gagtttgaag aatgggaaaa aggagaacat tgatcgtgag tattctgaga
     4561 ctgacatcct tgagagcgat gtccgtgaca agaaaaactt cgccgtgttg aaaaagatct
     4621 atctgaatga aaacgtgaac tcggaggaat tggaagtttt tcgtaacgac attaagaaga
     4681 aactgaacaa aatcaacagc ctgaagtact catttgaaaa gaataaggcg aattatcaaa
     4741 agattaatga gaataacatc gagaaggttg aaggtaagtc aaagcgtaac attatttacg
     4801 attattatcg tgagtcagcg aaacgtgacg cttatgtaag caatgtgaaa gaagcctttg
     4861 ataagcttta caaggaagag gacattgcaa aacttgttct tgaaattgag aaccttacga
     4921 agttagagaa atacaagatt cgcgagttct accacgaaat tattggacgt aagaatgaca
     4981 aggaaaactt tgcaaaaatc atctacgaag aaatccagaa tgttaataac atgaaagagt
     5041 tgatcgagaa ggtaccggac atgagtgagt tgaaaaagag ccaagtattt tacaagtatt
     5101 acttagacaa agaagagttg aacgacaaga acatcaaata cgcgttttgt catttcgtgg
     5161 aaatcgaaat gagtcagttg ctgaagaact acgtatataa gcgcttaagt aatatctcga
     5221 atgacaaaat taagcgtatc tttgaatacc agaacttgaa aaaattgatc gaaaataagc
     5281 tgttaaacaa acttgacacg tacgtccgta attgtggaaa gtataattat tatttgcaag
     5341 acggcgaaat tgccacttca gatttcatcg cccgcaaccg tcagaatgaa gcgtttcttc
     5401 gcaacatcat tggggtgtca tctgtggcct acttttctct tcgcaacatt cttgaaacgg
     5461 agaacgagaa tgatattact gggcgtatgc gcggcaaaac agttaagaac aataaaggtg
     5521 aagagaagta cgtgtccgga gaagttgata agatctataa tgaaaataag aagaacgagg
     5581 ttaaggagaa cttaaaaatg ttctattcgt acgatttcaa tatggacaac aagaatgaaa
     5641 tcgaagattt cttcgccaac atcgacgagg cgatttcttc catccgtcac ggtattgtcc
     5701 acttcaactt ggaattagaa ggtaaggata tctttgcgtt caagaacatt gcgccatccg
     5761 aaatctcaaa gaagatgttt cagaatgaga ttaacgagaa aaaactgaaa ttgaagatct
     5821 ttcgtcaact gaactctgcc aacgtgttcc gctatctcga aaagtataaa attctgaatt
     5881 accttaaacg tacacgcttc gagtttgtca ataaaaatat cccattcgtc ccgtctttca
     5941 ccaaattata ttcgcgcatt gatgacctga agaatagtct tgggatttac tggaaaactc
     6001 cgaaaacaaa cgacgacaat aagactaagg agattattga tgcccaaatc tatttgctta
     6061 aaaacatcta ttacggggag ttcctgaatt atttcatgtc gaacaatggt aatttctttg
     6121 agatttctaa agaaatcatc gaattgaaca agaacgataa acgcaactta aagactgggt
     6181 tttacaagct gcaaaagttt gaagacatcc aggagaagat tccaaaggaa tacttggcga
     6241 atatccagtc cctgtacatg attaatgccg gtaatcagga cgaagaagaa aaggacactt
     6301 atattgattt cattcaaaag atcttcttaa agggatttat gacgtatctt gctaataacg
     6361 gtcgtttaag tctgatttac atcggctcgg atgaagaaac aaatacgtca ttagcagaaa
     6421 agaagcaaga gtttgacaag ttcttgaaga agtacgagca gaacaataat atcaagatcc
     6481 cctatgagat caatgaattc ctgcgtgaga tcaaactggg aaacatcctg aagtatactg
     6541 agcgtttaaa catgttctac cttatcttaa agcttttgaa tcacaaggag ctgacaaatc
     6601 tgaagggtag tcttgaaaaa tatcagtctg ccaataagga agaagcgttc tctgaccaat
     6661 tggagttaat taacctgctt aaccttgaca acaaccgcgt gacggaagac ttcgaattag
     6721 aggccgacga gattggaaaa tttcttgatt tcaatggcaa caaagttaag gataacaagg
     6781 aactgaaaaa gttcgataca aacaagatct actttgacgg cgagaacatt atcaaacacc
     6841 gtgccttcta caatattaag aaatatggca tgttaaactt actggagaaa attgccgaca
     6901 aggctggata caagatctcg atcgaagagc tgaagaaata ctccaataaa aagaatgaga
     6961 tcgagaagaa ccataagatg caggaaaatc tgcaccgcaa atacgctcgt ccccgtaaag
     7021 acgagaagtt tacagatgag gactatgaaa gttacaagca agctattgag aatattgagg
     7081 agtacaccca ccttaagaac aaggtagaat tcaatgagct gaatttactg cagggcctgt
     7141 tgctgcgcat tttacatcgt ttagtcggat atacctcaat ttgggaacgc gatctgcgct
     7201 tccgccttaa aggtgagttc ccagaaaacc aatacatcga agagatcttc aactttgaaa
     7261 ataagaagaa cgtgaagtac aaagggggtc agattgtaga gaaatacatt aaattctaca
     7321 aggaattaca tcaaaatgat gaagttaaga tcaacaagta cagttccgcg aatatcaagg
     7381 tgttgaagca agaaaagaag gacctttata ttcgcaatta catcgcccac ttcaattata
     7441 ttcctcacgc cgagatctca ctgctggaag tccttgaaaa tttgcgtaaa ttgctgtcct
     7501 acgatcgcgc actgaaaaat gccgtaatga aatcagtagt tgatatcctt aaggagtatg
     7561 gttttgtagc cacattcaaa atcggggcgg acaagaagat cggtattcag acactggaga
     7621 gcgaaaaaat cgtgcatctt aagaatctta agaagaagaa gttaatgact gaccgcaatt
     7681 ccgaggaact ttgcaaattg gtgaagatta tgtttgaata caaaatggaa gagaaaaagt
     7741 ctgaaaacta ataacattgg aagtggataa cggatccgcg atcgcggcgc gccacctggt
     7801 ggccggccgg taccacgcgt gcgcgctgat ccggctgcta acaaagcccg aaaggaagct
     7861 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     7921 gtcttgaggg gttttttgct gaaaggagga actatatccg gatatccaca ggacgggtgt
     7981 ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg
     8041 gcggccaaag cggtcggaca gtgctccgag aacgggtgcg catagaaatt gcatcaacgc
     8101 atatagcgct agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg
     8161 caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt
     8221 gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca
     8281 atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga
     8341 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     8401 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     8461 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     8521 tgcttcaata acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     8581 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     8641 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     8701 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     8761 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     8821 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     8881 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     8941 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     9001 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     9061 taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac
     9121 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     9181 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     9241 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     9301 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     9361 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     9421 aagtttactc atat
//
