LOCUS       sequence-181377-        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_R1079A_K1080A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    403..542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(468..487)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     628..650
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(644..835)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2293..2312
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2396..2415
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2396..2414
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2503..2525
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2545..2562
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2572..3672
                     /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     3646..3669
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3727..3747
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7359..7377)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7373..7420
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7786..7791,7792..7808,7809..7814))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7841..7859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7927..8031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8032..8100,8101..8892)
                     /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(8250..8269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9063..9434,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
      421 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
      481 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
      541 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
      601 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
      661 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
      721 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
      781 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
      841 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
      901 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
      961 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     1021 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     1081 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     1141 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     1201 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     1261 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc
     1321 ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag
     1381 ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg
     1441 tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc
     1501 accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa
     1561 cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat
     1621 cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc
     1681 tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag
     1741 aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag
     1801 cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     1861 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1921 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1981 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     2041 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgacg
     2101 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     2161 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     2221 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     2281 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     2341 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     2401 cgactcacta tagggagacc acaacggttt ccctctagtg ccggctccgg agagctcttt
     2461 aattaagcgg ccgccctgca ggactcgagt tctagaaata attttgttta actttaagaa
     2521 ggagatatac atatgaaatc ttctcaccat caccatcacc atggttcttc tatgaaaatc
     2581 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
     2641 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
     2701 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
     2761 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
     2821 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
     2881 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
     2941 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
     3001 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
     3061 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
     3121 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
     3181 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
     3241 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
     3301 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
     3361 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     3421 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     3481 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa
     3541 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     3601 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     3661 gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg
     3721 atcgaggaaa acctgtactt ccaatccaat gcaatgaaag tgacgaaggt aggaggcatt
     3781 tcgcataaga agtacacgtc cgaaggccgc ttagtgaagt cagaatcgga agaaaatcgc
     3841 acagacgaac gtctgtcggc gttgcttaat atgcgccttg acatgtatat caagaatccc
     3901 agcagcacgg aaaccaagga aaatcaaaaa cgcattggga aattaaagaa attcttctca
     3961 aacaaaatgg tctatcttaa agacaatacc ttgagtttga agaatgggaa aaaggagaac
     4021 attgatcgtg agtattctga gactgacatc cttgagagcg atgtccgtga caagaaaaac
     4081 ttcgccgtgt tgaaaaagat ctatctgaat gaaaacgtga actcggagga attggaagtt
     4141 tttcgtaacg acattaagaa gaaactgaac aaaatcaaca gcctgaagta ctcatttgaa
     4201 aagaataagg cgaattatca aaagattaat gagaataaca tcgagaaggt tgaaggtaag
     4261 tcaaagcgta acattattta cgattattat cgtgagtcag cgaaacgtga cgcttatgta
     4321 agcaatgtga aagaagcctt tgataagctt tacaaggaag aggacattgc aaaacttgtt
     4381 cttgaaattg agaaccttac gaagttagag aaatacaaga ttcgcgagtt ctaccacgaa
     4441 attattggac gtaagaatga caaggaaaac tttgcaaaaa tcatctacga agaaatccag
     4501 aatgttaata acatgaaaga gttgatcgag aaggtaccgg acatgagtga gttgaaaaag
     4561 agccaagtat tttacaagta ttacttagac aaagaagagt tgaacgacaa gaacatcaaa
     4621 tacgcgtttt gtcatttcgt ggaaatcgaa atgagtcagt tgctgaagaa ctacgtatat
     4681 aagcgcttaa gtaatatctc gaatgacaaa attaagcgta tctttgaata ccagaacttg
     4741 aaaaaattga tcgaaaataa gctgttaaac aaacttgaca cgtacgtccg taattgtgga
     4801 aagtataatt attatttgca agacggcgaa attgccactt cagatttcat cgcccgcaac
     4861 cgtcagaatg aagcgtttct tcgcaacatc attggggtgt catctgtggc ctacttttct
     4921 cttcgcaaca ttcttgaaac ggagaacgag aatgatatta ctgggcgtat gcgcggcaaa
     4981 acagttaaga acaataaagg tgaagagaag tacgtgtccg gagaagttga taagatctat
     5041 aatgaaaata agaagaacga ggttaaggag aacttaaaaa tgttctattc gtacgatttc
     5101 aatatggaca acaagaatga aatcgaagat ttcttcgcca acatcgacga ggcgatttct
     5161 tccatccgtc acggtattgt ccacttcaac ttggaattag aaggtaagga tatctttgcg
     5221 ttcaagaaca ttgcgccatc cgaaatctca aagaagatgt ttcagaatga gattaacgag
     5281 aaaaaactga aattgaagat ctttcgtcaa ctgaactctg ccaacgtgtt ccgctatctc
     5341 gaaaagtata aaattctgaa ttaccttaaa cgtacacgct tcgagtttgt caataaaaat
     5401 atcccattcg tcccgtcttt caccaaatta tattcgcgca ttgatgacct gaagaatagt
     5461 cttgggattt actggaaaac tccgaaaaca aacgacgaca ataagactaa ggagattatt
     5521 gatgcccaaa tctatttgct taaaaacatc tattacgggg agttcctgaa ttatttcatg
     5581 tcgaacaatg gtaatttctt tgagatttct aaagaaatca tcgaattgaa caagaacgat
     5641 aaacgcaact taaagactgg gttttacaag ctgcaaaagt ttgaagacat ccaggagaag
     5701 attccaaagg aatacttggc gaatatccag tccctgtaca tgattaatgc cggtaatcag
     5761 gacgaagaag aaaaggacac ttatattgat ttcattcaaa agatcttctt aaagggattt
     5821 atgacgtatc ttgctaataa cggtcgttta agtctgattt acatcggctc ggatgaagaa
     5881 acaaatacgt cattagcaga aaagaagcaa gagtttgaca agttcttgaa gaagtacgag
     5941 cagaacaata atatcaagat cccctatgag atcaatgaat tcctgcgtga gatcaaactg
     6001 ggaaacatcc tgaagtatac tgagcgttta aacatgttct accttatctt aaagcttttg
     6061 aatcacaagg agctgacaaa tctgaagggt agtcttgaaa aatatcagtc tgccaataag
     6121 gaagaagcgt tctctgacca attggagtta attaacctgc ttaaccttga caacaaccgc
     6181 gtgacggaag acttcgaatt agaggccgac gagattggaa aatttcttga tttcaatggc
     6241 aacaaagtta aggataacaa ggaactgaaa aagttcgata caaacaagat ctactttgac
     6301 ggcgagaaca ttatcaaaca ccgtgccttc tacaatatta agaaatatgg catgttaaac
     6361 ttactggaga aaattgccga caaggctgga tacaagatct cgatcgaaga gctgaagaaa
     6421 tactccaata aaaagaatga gatcgagaag aaccataaga tgcaggaaaa tctgcaccgc
     6481 aaatacgctc gtccccgtaa agacgagaag tttacagatg aggactatga aagttacaag
     6541 caagctattg agaatattga ggagtacacc caccttaaga acaaggtaga attcaatgag
     6601 ctgaatttac tgcagggcct gttgctgcgc attttacatc gtttagtcgg atatacctca
     6661 atttgggaac gcgatctgcg cttccgcctt aaaggtgagt tcccagaaaa ccaatacatc
     6721 gaagagatct tcaactttga aaataagaag aacgtgaagt acaaaggggg tcagattgta
     6781 gagaaataca ttaaattcta caaggaatta catcaaaatg atgaagttaa gatcaacaag
     6841 tacagttccg cgaatatcaa ggtgttgaag caagaaaaga aggaccttta tattcgcaat
     6901 tacatcgccc acttcaatta tattcctcac gccgagatct cactgctgga agtccttgaa
     6961 aatttgcgta aattgctgtc ctacgatgcc gcactgaaaa atgccgtaat gaaatcagta
     7021 gttgatatcc ttaaggagta tggttttgta gccacattca aaatcggggc ggacaagaag
     7081 atcggtattc agacactgga gagcgaaaaa atcgtgcatc ttaagaatct taagaagaag
     7141 aagttaatga ctgaccgcaa ttccgaggaa ctttgcaaat tggtgaagat tatgtttgaa
     7201 tacaaaatgg aagagaaaaa gtctgaaaac taataacatt ggaagtggat aacggatccg
     7261 cgatcgcggc gcgccacctg gtggccggcc ggtaccacgc gtgcgcgctg atccggctgc
     7321 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     7381 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     7441 cggatatcca caggacgggt gtggtcgcca tgatcgcgta gtcgatagtg gctccaagta
     7501 gcgaagcgag caggactggg cggcggccaa agcggtcgga cagtgctccg agaacgggtg
     7561 cgcatagaaa ttgcatcaac gcatatagcg ctagcagcac gccatagtga ctggcgatgc
     7621 tgtcggaatg gacgatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc
     7681 ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac
     7741 acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa agcttatcga
     7801 tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt
     7861 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     7921 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7981 atgagacaat aaccctgata aatgcttcaa taacattgaa aaaggaagag tatgagtatt
     8041 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     8101 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     8161 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     8221 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     8281 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     8341 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     8401 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     8461 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     8521 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     8581 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     8641 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     8701 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     8761 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     8821 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     8881 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     8941 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     9001 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     9061 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     9121 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     9181 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     9241 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     9301 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     9361 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     9421 acctacaccg aact
//
