LOCUS       sequence-181520-        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_K1087A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1643..1662
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1746..1765
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1746..1764
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1853..1875
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1895..1912
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1922..3022
                     /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     2996..3019
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3077..3097
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6709..6727)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6723..6770
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7136..7141,7142..7158,7159..7164))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7191..7209)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7277..7381
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7382..7450,7451..8242)
                     /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(7600..7619)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8413..9001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8902..8921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9155..9172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9187..9326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9252..9271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9412..9434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(9428..9434,1..185))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
       61 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
      121 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
      181 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
      241 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
      301 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
      361 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
      421 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
      481 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
      541 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
      601 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
      661 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
      721 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
      781 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
      841 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
      901 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
      961 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     1021 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     1081 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     1141 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     1201 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     1261 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     1321 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     1381 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     1441 tcggtcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     1501 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     1561 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     1621 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     1681 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     1741 gaaattaata cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg
     1801 agagctcttt aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta
     1861 actttaagaa ggagatatac atatgaaatc ttctcaccat caccatcacc atggttcttc
     1921 tatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
     1981 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
     2041 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
     2101 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
     2161 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
     2221 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
     2281 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
     2341 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
     2401 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
     2461 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
     2521 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
     2581 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
     2641 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
     2701 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     2761 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     2821 gggtgccgta gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc
     2881 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     2941 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     3001 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     3061 caacctcggg atcgaggaaa acctgtactt ccaatccaat gcaatgaaag tgacgaaggt
     3121 aggaggcatt tcgcataaga agtacacgtc cgaaggccgc ttagtgaagt cagaatcgga
     3181 agaaaatcgc acagacgaac gtctgtcggc gttgcttaat atgcgccttg acatgtatat
     3241 caagaatccc agcagcacgg aaaccaagga aaatcaaaaa cgcattggga aattaaagaa
     3301 attcttctca aacaaaatgg tctatcttaa agacaatacc ttgagtttga agaatgggaa
     3361 aaaggagaac attgatcgtg agtattctga gactgacatc cttgagagcg atgtccgtga
     3421 caagaaaaac ttcgccgtgt tgaaaaagat ctatctgaat gaaaacgtga actcggagga
     3481 attggaagtt tttcgtaacg acattaagaa gaaactgaac aaaatcaaca gcctgaagta
     3541 ctcatttgaa aagaataagg cgaattatca aaagattaat gagaataaca tcgagaaggt
     3601 tgaaggtaag tcaaagcgta acattattta cgattattat cgtgagtcag cgaaacgtga
     3661 cgcttatgta agcaatgtga aagaagcctt tgataagctt tacaaggaag aggacattgc
     3721 aaaacttgtt cttgaaattg agaaccttac gaagttagag aaatacaaga ttcgcgagtt
     3781 ctaccacgaa attattggac gtaagaatga caaggaaaac tttgcaaaaa tcatctacga
     3841 agaaatccag aatgttaata acatgaaaga gttgatcgag aaggtaccgg acatgagtga
     3901 gttgaaaaag agccaagtat tttacaagta ttacttagac aaagaagagt tgaacgacaa
     3961 gaacatcaaa tacgcgtttt gtcatttcgt ggaaatcgaa atgagtcagt tgctgaagaa
     4021 ctacgtatat aagcgcttaa gtaatatctc gaatgacaaa attaagcgta tctttgaata
     4081 ccagaacttg aaaaaattga tcgaaaataa gctgttaaac aaacttgaca cgtacgtccg
     4141 taattgtgga aagtataatt attatttgca agacggcgaa attgccactt cagatttcat
     4201 cgcccgcaac cgtcagaatg aagcgtttct tcgcaacatc attggggtgt catctgtggc
     4261 ctacttttct cttcgcaaca ttcttgaaac ggagaacgag aatgatatta ctgggcgtat
     4321 gcgcggcaaa acagttaaga acaataaagg tgaagagaag tacgtgtccg gagaagttga
     4381 taagatctat aatgaaaata agaagaacga ggttaaggag aacttaaaaa tgttctattc
     4441 gtacgatttc aatatggaca acaagaatga aatcgaagat ttcttcgcca acatcgacga
     4501 ggcgatttct tccatccgtc acggtattgt ccacttcaac ttggaattag aaggtaagga
     4561 tatctttgcg ttcaagaaca ttgcgccatc cgaaatctca aagaagatgt ttcagaatga
     4621 gattaacgag aaaaaactga aattgaagat ctttcgtcaa ctgaactctg ccaacgtgtt
     4681 ccgctatctc gaaaagtata aaattctgaa ttaccttaaa cgtacacgct tcgagtttgt
     4741 caataaaaat atcccattcg tcccgtcttt caccaaatta tattcgcgca ttgatgacct
     4801 gaagaatagt cttgggattt actggaaaac tccgaaaaca aacgacgaca ataagactaa
     4861 ggagattatt gatgcccaaa tctatttgct taaaaacatc tattacgggg agttcctgaa
     4921 ttatttcatg tcgaacaatg gtaatttctt tgagatttct aaagaaatca tcgaattgaa
     4981 caagaacgat aaacgcaact taaagactgg gttttacaag ctgcaaaagt ttgaagacat
     5041 ccaggagaag attccaaagg aatacttggc gaatatccag tccctgtaca tgattaatgc
     5101 cggtaatcag gacgaagaag aaaaggacac ttatattgat ttcattcaaa agatcttctt
     5161 aaagggattt atgacgtatc ttgctaataa cggtcgttta agtctgattt acatcggctc
     5221 ggatgaagaa acaaatacgt cattagcaga aaagaagcaa gagtttgaca agttcttgaa
     5281 gaagtacgag cagaacaata atatcaagat cccctatgag atcaatgaat tcctgcgtga
     5341 gatcaaactg ggaaacatcc tgaagtatac tgagcgttta aacatgttct accttatctt
     5401 aaagcttttg aatcacaagg agctgacaaa tctgaagggt agtcttgaaa aatatcagtc
     5461 tgccaataag gaagaagcgt tctctgacca attggagtta attaacctgc ttaaccttga
     5521 caacaaccgc gtgacggaag acttcgaatt agaggccgac gagattggaa aatttcttga
     5581 tttcaatggc aacaaagtta aggataacaa ggaactgaaa aagttcgata caaacaagat
     5641 ctactttgac ggcgagaaca ttatcaaaca ccgtgccttc tacaatatta agaaatatgg
     5701 catgttaaac ttactggaga aaattgccga caaggctgga tacaagatct cgatcgaaga
     5761 gctgaagaaa tactccaata aaaagaatga gatcgagaag aaccataaga tgcaggaaaa
     5821 tctgcaccgc aaatacgctc gtccccgtaa agacgagaag tttacagatg aggactatga
     5881 aagttacaag caagctattg agaatattga ggagtacacc caccttaaga acaaggtaga
     5941 attcaatgag ctgaatttac tgcagggcct gttgctgcgc attttacatc gtttagtcgg
     6001 atatacctca atttgggaac gcgatctgcg cttccgcctt aaaggtgagt tcccagaaaa
     6061 ccaatacatc gaagagatct tcaactttga aaataagaag aacgtgaagt acaaaggggg
     6121 tcagattgta gagaaataca ttaaattcta caaggaatta catcaaaatg atgaagttaa
     6181 gatcaacaag tacagttccg cgaatatcaa ggtgttgaag caagaaaaga aggaccttta
     6241 tattcgcaat tacatcgccc acttcaatta tattcctcac gccgagatct cactgctgga
     6301 agtccttgaa aatttgcgta aattgctgtc ctacgatcgc aaactgaaaa atgccgtaat
     6361 ggcatcagta gttgatatcc ttaaggagta tggttttgta gccacattca aaatcggggc
     6421 ggacaagaag atcggtattc agacactgga gagcgaaaaa atcgtgcatc ttaagaatct
     6481 taagaagaag aagttaatga ctgaccgcaa ttccgaggaa ctttgcaaat tggtgaagat
     6541 tatgtttgaa tacaaaatgg aagagaaaaa gtctgaaaac taataacatt ggaagtggat
     6601 aacggatccg cgatcgcggc gcgccacctg gtggccggcc ggtaccacgc gtgcgcgctg
     6661 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     6721 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     6781 gaactatatc cggatatcca caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg
     6841 gctccaagta gcgaagcgag caggactggg cggcggccaa agcggtcgga cagtgctccg
     6901 agaacgggtg cgcatagaaa ttgcatcaac gcatatagcg ctagcagcac gccatagtga
     6961 ctggcgatgc tgtcggaatg gacgatatcc cgcaagaggc ccggcagtac cggcataacc
     7021 aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta
     7081 gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa
     7141 agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat
     7201 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     7261 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     7321 gtatccgctc atgagacaat aaccctgata aatgcttcaa taacattgaa aaaggaagag
     7381 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     7441 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     7501 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     7561 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     7621 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     7681 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     7741 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7801 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     7861 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     7921 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7981 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     8041 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     8101 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     8161 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     8221 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     8281 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     8341 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     8401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     8461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     8521 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     8581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     8641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     8701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     8761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     8941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     9001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     9061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     9121 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     9181 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     9241 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     9301 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     9361 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     9421 gcatgtgtca gagg
//
