LOCUS       sequence-181512-        9110 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 9110)
  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 9110)
  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-Lne_Cas13a_WT
FEATURES             Location/Qualifiers
     source          1..9110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1565..1584
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1668..1687
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1668..1686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1775..1797
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1817..1834
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1844..2944
                     /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     2918..2941
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2999..3019
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6307..6325)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6321..6368
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(6734..6739,6740..6756,6757..6762))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6789..6807)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6875..6979
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6980..7048,7049..7840)
                     /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(7198..7217)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8011..8599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8500..8519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8753..8770
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8785..8924
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8850..8869)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9010..9032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(join(9026..9110,1..107))
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
       61 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
      121 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
      181 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
      241 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
      301 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
      361 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
      421 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
      481 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
      541 ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg
      601 cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt
      661 tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg
      721 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
      781 gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
      841 atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg atcgaagtta
      901 ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc
      961 tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa
     1021 tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg
     1081 ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga
     1141 cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg
     1201 tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc
     1261 ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg
     1321 cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga cgctctccct
     1381 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     1441 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     1501 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     1561 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     1621 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     1681 tatagggaga ccacaacggt ttccctctag tgccggctcc ggagagctct ttaattaagc
     1741 ggccgccctg caggactcga gttctagaaa taattttgtt taactttaag aaggagatat
     1801 acatatgaaa tcttctcacc atcaccatca ccatggttct tctatgaaaa tcgaagaagg
     1861 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
     1921 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
     1981 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
     2041 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
     2101 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
     2161 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
     2221 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
     2281 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
     2341 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
     2401 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
     2461 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
     2521 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
     2581 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     2641 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     2701 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     2761 gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca
     2821 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     2881 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     2941 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgagga
     3001 aaacctgtac ttccaatcca atgcaatgaa aattaccaag atgcgcgtgg atggtcgtac
     3061 gattgtgatg gaacgtactt cgaaagaagg acagctgggc tatgaaggca tcgacgggaa
     3121 caaaacgact gagattattt tcgataaaaa gaaggaatct ttttacaaga gcattttgaa
     3181 caaaaccgtc cgcaaaccgg acgagaaaga gaaaaatcgc cgcaaacaag caattaacaa
     3241 agcaattaat aaggagatca cggagttaat gctggccgtg cttcatcaag aggtgccttc
     3301 gcaaaaactg cacaacttaa agtcacttaa cacggagtcg ttaacgaaat tattcaagcc
     3361 taagttccag aatatgatta gttaccctcc ttccaaaggc gctgagcatg tgcagttttg
     3421 cttaacagat attgcggttc cggcaatccg tgatcttgat gagattaagc cggattgggg
     3481 tatctttttt gaaaaactta agccttacac cgattgggct gagagctaca tccattacaa
     3541 acaaacaacc attcaaaagt cgattgagca gaacaagatt caatcaccgg actccccgcg
     3601 caagttggta ttgcaaaaat atgtcactgc cttcctgaat ggtgagcccc ttggattaga
     3661 tttggtcgct aagaagtata aattagccga cctggctgag agtttcaaac tggtcgacct
     3721 taatgaggat aaatcagcga attacaaaat taaagcctgt cttcagcagc accagcgtaa
     3781 catcttggat gagttaaagg aagatccaga acttaaccaa tatggtattg aagtcaaaaa
     3841 gtacattcag cgttacttcc ctattaaacg tgcaccaaat cgctccaaac atgcacgtgc
     3901 agacttctta aagaaagaat taattgaatc gacagtggaa caacaattca aaaacgcggt
     3961 ctatcactac gtattggaac agggaaaaat ggaagcatac gaacttactg accctaaaac
     4021 taaagatctt caggacattc gcagcggaga agcattctca tttaaattca tcaacgcgtg
     4081 cgcattcgca agcaacaact taaagatgat tcttaatccg gagtgcgaaa aagacattct
     4141 gggcaaaggc aacttcaaaa aaaacctgcc caattcaacg acgcgttctg acgtcgtaaa
     4201 aaaaatgatt ccttttttca gtgacgaact gcaaaacgtg aattttgacg aggccatctg
     4261 ggccatccgt ggctcaatcc aacaaatccg taatgaagtg tatcactgta agaagcattc
     4321 ttggaaatcg atccttaaga ttaaaggctt tgaatttgaa ccgaataaca tgaaatacgc
     4381 agattccgat atgcaaaagc ttatggacaa agacatcgcc aagattccgg agtttattga
     4441 ggaaaaattg aaatcatcag gcgtagtgcg tttttaccgt catgacgagc tgcaaagcat
     4501 ttgggaaatg aagcaaggtt tctcgctgct tactacgaac gcgccgttcg tgccctcgtt
     4561 taaacgcgtg tatgcgaagg ggcatgatta tcaaacaagc aagaaccgct actacaatct
     4621 ggaccttaca accttcgaca tcttggaata cggagaagaa gactttcgtg ctcgctattt
     4681 cttaactaag cttgtatact atcaacagtt catgccttgg tttactgccg ataataacgc
     4741 ctttcgcgac gcagccaatt ttgtgttacg cttaaataaa aatcgtcagc aggatgcgaa
     4801 agcattcatc aacatccgcg aagttgaaga gggagagatg ccgcgcgatt acatgggtta
     4861 tgttcagggc caaatcgcga tccatgaaga ttcgattgag gatactccta accatttcga
     4921 aaagtttatc agtcaagttt ttattaaagg ttttgaccgt cacatgcgtt ccgcgaattt
     4981 gaaattcatc aagaatccac gcaatcaagg ccttgaacag tcggagattg aagaaatgtc
     5041 gttcgacatc aaggtagagc cttcctttct gaaaaacaag gatgactaca ttgctttttg
     5101 gattttttgt aaaatgctgg atgcccgcca cttatcagaa ttacgtaatg agatgattaa
     5161 gtatgatgga cacttgacgg gagaacaaga gatcattggt ttagcgttac tgggtgttga
     5221 cagccgcgag aacgactgga agcagttttt ttcctcagag cgcgaatatg agaaaatcat
     5281 gaagggctac gtcgtggaag aactttatca gcgcgagcca tatcgtcagt cagatgggaa
     5341 gacgcctatc ttattccgtg gcgtggaaca ggcacgcaaa tatggtacag aaaccgtgat
     5401 tcaacgcctt tttgacgcca atcccgaatt taaggtcagc aaatgtaact tagccgagtg
     5461 ggagcgccaa aaagagacca ttgaagagac aatcaaacgc cgcaaggaat tgcacaatga
     5521 atgggccaag aaccctaaga aaccgcagaa caatgcgttc ttcaaagagt ataaagaatg
     5581 ctgcgatgct atcgatgcct acaattggca taagaataaa accacattgg cctatgtaaa
     5641 tgaactgcat cacttactta tcgaaatctt ggggcgctat gttggctatg ttgcgatcgc
     5701 ggatcgcgat ttccagtgta tggccaatca atactttaag cactcaggca ttaccgaacg
     5761 tgtcgaatac tggggagaca atcgccttaa aagcattaaa aagttggaca cttttctgaa
     5821 gaaagaaggt ctttttgtaa gtgagaagaa cgcccgcaac catattgcgc atttaaacta
     5881 cctgtcatta aagtctgagt gtactctttt atacctttcc gagcgcttac gtgagatctt
     5941 caaatacgat cgtaagctta aaaacgcagt ttcaaaatcc ttaattgaca ttcttgatcg
     6001 ccatgggatg tcagttgtct ttgccaacct taaagaaaac aaacaccgcc tggttatcaa
     6061 gagtttagaa ccaaagaaac ttcgtcattt aggtgggaag aaaatcgatg gcggatacat
     6121 tgagactaac caagtaagcg aggagtattg cggaatcgtg aaacgtttgc ttgaaatgta
     6181 ataacattgg aagtggataa cggatccgcg atcgcggcgc gccacctggt ggccggccgg
     6241 taccacgcgt gcgcgctgat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     6301 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     6361 gttttttgct gaaaggagga actatatccg gatatccaca ggacgggtgt ggtcgccatg
     6421 atcgcgtagt cgatagtggc tccaagtagc gaagcgagca ggactgggcg gcggccaaag
     6481 cggtcggaca gtgctccgag aacgggtgcg catagaaatt gcatcaacgc atatagcgct
     6541 agcagcacgc catagtgact ggcgatgctg tcggaatgga cgatatcccg caagaggccc
     6601 ggcagtaccg gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg
     6661 acgatgagcg cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt
     6721 gataaactac cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag
     6781 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     6841 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     6901 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6961 acattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     7021 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     7081 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     7141 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     7201 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     7261 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     7321 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     7381 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     7441 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     7501 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     7561 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     7621 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     7681 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     7741 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     7801 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     7861 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     7921 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7981 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     8041 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     8101 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     8161 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     8221 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     8281 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     8341 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     8401 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     8461 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     8521 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     8581 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     8641 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     8701 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     8761 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     8821 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     8881 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     8941 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     9001 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     9061 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
//
