LOCUS       sequence-181383-        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_N314A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     628..651
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             709..729
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(4341..4359)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4355..4402
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4768..4773,4774..4790,4791..4796))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4823..4841)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4909..5013
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5014..5082,5083..5874)
                     /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(5232..5251)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6045..6633
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6534..6553
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6787..6804
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6819..6958
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6884..6903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7044..7066
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7060..7251)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     8709..8728
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     8812..8831
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8812..8830
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             8919..8941
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             8961..8978
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(8988..9434,1..654)
                     /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"
ORIGIN
        1 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
       61 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
      121 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
      181 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
      241 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
      301 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
      361 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
      421 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
      481 gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa
      541 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
      601 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
      661 agctcgaaca acaacaacaa taacaataac aacaacctcg ggatcgagga aaacctgtac
      721 ttccaatcca atgcaatgaa agtgacgaag gtaggaggca tttcgcataa gaagtacacg
      781 tccgaaggcc gcttagtgaa gtcagaatcg gaagaaaatc gcacagacga acgtctgtcg
      841 gcgttgctta atatgcgcct tgacatgtat atcaagaatc ccagcagcac ggaaaccaag
      901 gaaaatcaaa aacgcattgg gaaattaaag aaattcttct caaacaaaat ggtctatctt
      961 aaagacaata ccttgagttt gaagaatggg aaaaaggaga acattgatcg tgagtattct
     1021 gagactgaca tccttgagag cgatgtccgt gacaagaaaa acttcgccgt gttgaaaaag
     1081 atctatctga atgaaaacgt gaactcggag gaattggaag tttttcgtaa cgacattaag
     1141 aagaaactga acaaaatcaa cagcctgaag tactcatttg aaaagaataa ggcgaattat
     1201 caaaagatta atgagaataa catcgagaag gttgaaggta agtcaaagcg taacattatt
     1261 tacgattatt atcgtgagtc agcgaaacgt gacgcttatg taagcaatgt gaaagaagcc
     1321 tttgataagc tttacaagga agaggacatt gcaaaacttg ttcttgaaat tgagaacctt
     1381 acgaagttag agaaatacaa gattcgcgag ttctaccacg aaattattgg acgtaagaat
     1441 gacaaggaaa actttgcaaa aatcatctac gaagaaatcc agaatgttaa taacatgaaa
     1501 gagttgatcg agaaggtacc ggacatgagt gagttgaaaa agagccaagt attttacaag
     1561 tattacttag acaaagaaga gttgaacgac aagaacatca aatacgcgtt ttgtcatttc
     1621 gtggaaatcg aaatgagtca gttgctgaag aactacgtat ataagcgctt aagtgcaatc
     1681 tcgaatgaca aaattaagcg tatctttgaa taccagaact tgaaaaaatt gatcgaaaat
     1741 aagctgttaa acaaacttga cacgtacgtc cgtaattgtg gaaagtataa ttattatttg
     1801 caagacggcg aaattgccac ttcagatttc atcgcccgca accgtcagaa tgaagcgttt
     1861 cttcgcaaca tcattggggt gtcatctgtg gcctactttt ctcttcgcaa cattcttgaa
     1921 acggagaacg agaatgatat tactgggcgt atgcgcggca aaacagttaa gaacaataaa
     1981 ggtgaagaga agtacgtgtc cggagaagtt gataagatct ataatgaaaa taagaagaac
     2041 gaggttaagg agaacttaaa aatgttctat tcgtacgatt tcaatatgga caacaagaat
     2101 gaaatcgaag atttcttcgc caacatcgac gaggcgattt cttccatccg tcacggtatt
     2161 gtccacttca acttggaatt agaaggtaag gatatctttg cgttcaagaa cattgcgcca
     2221 tccgaaatct caaagaagat gtttcagaat gagattaacg agaaaaaact gaaattgaag
     2281 atctttcgtc aactgaactc tgccaacgtg ttccgctatc tcgaaaagta taaaattctg
     2341 aattacctta aacgtacacg cttcgagttt gtcaataaaa atatcccatt cgtcccgtct
     2401 ttcaccaaat tatattcgcg cattgatgac ctgaagaata gtcttgggat ttactggaaa
     2461 actccgaaaa caaacgacga caataagact aaggagatta ttgatgccca aatctatttg
     2521 cttaaaaaca tctattacgg ggagttcctg aattatttca tgtcgaacaa tggtaatttc
     2581 tttgagattt ctaaagaaat catcgaattg aacaagaacg ataaacgcaa cttaaagact
     2641 gggttttaca agctgcaaaa gtttgaagac atccaggaga agattccaaa ggaatacttg
     2701 gcgaatatcc agtccctgta catgattaat gccggtaatc aggacgaaga agaaaaggac
     2761 acttatattg atttcattca aaagatcttc ttaaagggat ttatgacgta tcttgctaat
     2821 aacggtcgtt taagtctgat ttacatcggc tcggatgaag aaacaaatac gtcattagca
     2881 gaaaagaagc aagagtttga caagttcttg aagaagtacg agcagaacaa taatatcaag
     2941 atcccctatg agatcaatga attcctgcgt gagatcaaac tgggaaacat cctgaagtat
     3001 actgagcgtt taaacatgtt ctaccttatc ttaaagcttt tgaatcacaa ggagctgaca
     3061 aatctgaagg gtagtcttga aaaatatcag tctgccaata aggaagaagc gttctctgac
     3121 caattggagt taattaacct gcttaacctt gacaacaacc gcgtgacgga agacttcgaa
     3181 ttagaggccg acgagattgg aaaatttctt gatttcaatg gcaacaaagt taaggataac
     3241 aaggaactga aaaagttcga tacaaacaag atctactttg acggcgagaa cattatcaaa
     3301 caccgtgcct tctacaatat taagaaatat ggcatgttaa acttactgga gaaaattgcc
     3361 gacaaggctg gatacaagat ctcgatcgaa gagctgaaga aatactccaa taaaaagaat
     3421 gagatcgaga agaaccataa gatgcaggaa aatctgcacc gcaaatacgc tcgtccccgt
     3481 aaagacgaga agtttacaga tgaggactat gaaagttaca agcaagctat tgagaatatt
     3541 gaggagtaca cccaccttaa gaacaaggta gaattcaatg agctgaattt actgcagggc
     3601 ctgttgctgc gcattttaca tcgtttagtc ggatatacct caatttggga acgcgatctg
     3661 cgcttccgcc ttaaaggtga gttcccagaa aaccaataca tcgaagagat cttcaacttt
     3721 gaaaataaga agaacgtgaa gtacaaaggg ggtcagattg tagagaaata cattaaattc
     3781 tacaaggaat tacatcaaaa tgatgaagtt aagatcaaca agtacagttc cgcgaatatc
     3841 aaggtgttga agcaagaaaa gaaggacctt tatattcgca attacatcgc ccacttcaat
     3901 tatattcctc acgccgagat ctcactgctg gaagtccttg aaaatttgcg taaattgctg
     3961 tcctacgatc gcaaactgaa aaatgccgta atgaaatcag tagttgatat ccttaaggag
     4021 tatggttttg tagccacatt caaaatcggg gcggacaaga agatcggtat tcagacactg
     4081 gagagcgaaa aaatcgtgca tcttaagaat cttaagaaga agaagttaat gactgaccgc
     4141 aattccgagg aactttgcaa attggtgaag attatgtttg aatacaaaat ggaagagaaa
     4201 aagtctgaaa actaataaca ttggaagtgg ataacggatc cgcgatcgcg gcgcgccacc
     4261 tggtggccgg ccggtaccac gcgtgcgcgc tgatccggct gctaacaaag cccgaaagga
     4321 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     4381 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg
     4441 gtgtggtcgc catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg
     4501 ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca
     4561 acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat
     4621 cccgcaagag gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga
     4681 cggtgccgag gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt
     4741 agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat
     4801 gagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4861 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4921 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4981 taaatgcttc aataacattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5041 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5101 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5161 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5221 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     5281 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5341 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5401 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5461 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5521 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     5581 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5641 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5701 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5761 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5821 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5881 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5941 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6001 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6061 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6121 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6181 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6241 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6301 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6361 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6421 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6481 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6541 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6601 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6661 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6721 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     6781 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     6841 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     6901 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     6961 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     7021 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     7081 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     7141 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     7201 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     7261 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     7321 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     7381 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     7441 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     7501 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     7561 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     7621 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     7681 ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga
     7741 gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt
     7801 ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa
     7861 tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg
     7921 cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg
     7981 ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa
     8041 gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc
     8101 tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc
     8161 ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg
     8221 tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     8281 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     8341 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     8401 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     8461 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgacgctct
     8521 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     8581 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     8641 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     8701 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     8761 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     8821 tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag ctctttaatt
     8881 aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt taagaaggag
     8941 atatacatat gaaatcttct caccatcacc atcaccatgg ttcttctatg aaaatcgaag
     9001 aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc gctgaagtcg
     9061 gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg gataaactgg
     9121 aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc ttctgggcac
     9181 acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc ccggacaaag
     9241 cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac ggcaagctga
     9301 ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat ctgctgccga
     9361 acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa gcgaaaggta
     9421 agagcgcgct gatg
//
