LOCUS       sequence-181513-        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_E299A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     45..67
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(61..252)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1710..1729
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1813..1832
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1813..1831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1920..1942
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1962..1979
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1989..3089
                     /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     3063..3086
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3144..3164
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(6776..6794)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6790..6837
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7203..7208,7209..7225,7226..7231))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7258..7276)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7344..7448
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7449..7517,7518..8309)
                     /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(7667..7686)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8480..9068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8969..8988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9222..9239
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    9254..9393
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(9319..9338)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
       61 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
      121 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
      181 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
      241 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
      301 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
      361 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
      421 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
      481 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
      541 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
      601 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
      661 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
      721 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
      781 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
      841 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
      901 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
      961 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     1021 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     1081 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     1141 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     1201 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     1261 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     1321 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     1381 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     1441 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     1501 aagggcatcg gtcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     1561 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     1621 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     1681 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     1741 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     1801 atcccgcgaa attaatacga ctcactatag ggagaccaca acggtttccc tctagtgccg
     1861 gctccggaga gctctttaat taagcggccg ccctgcagga ctcgagttct agaaataatt
     1921 ttgtttaact ttaagaagga gatatacata tgaaatcttc tcaccatcac catcaccatg
     1981 gttcttctat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     2041 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     2101 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     2161 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     2221 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     2281 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     2341 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     2401 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     2461 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     2521 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     2581 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     2641 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     2701 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     2761 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     2821 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     2881 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     2941 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     3001 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     3061 tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac aacaataaca
     3121 ataacaacaa cctcgggatc gaggaaaacc tgtacttcca atccaatgca atgaaagtga
     3181 cgaaggtagg aggcatttcg cataagaagt acacgtccga aggccgctta gtgaagtcag
     3241 aatcggaaga aaatcgcaca gacgaacgtc tgtcggcgtt gcttaatatg cgccttgaca
     3301 tgtatatcaa gaatcccagc agcacggaaa ccaaggaaaa tcaaaaacgc attgggaaat
     3361 taaagaaatt cttctcaaac aaaatggtct atcttaaaga caataccttg agtttgaaga
     3421 atgggaaaaa ggagaacatt gatcgtgagt attctgagac tgacatcctt gagagcgatg
     3481 tccgtgacaa gaaaaacttc gccgtgttga aaaagatcta tctgaatgaa aacgtgaact
     3541 cggaggaatt ggaagttttt cgtaacgaca ttaagaagaa actgaacaaa atcaacagcc
     3601 tgaagtactc atttgaaaag aataaggcga attatcaaaa gattaatgag aataacatcg
     3661 agaaggttga aggtaagtca aagcgtaaca ttatttacga ttattatcgt gagtcagcga
     3721 aacgtgacgc ttatgtaagc aatgtgaaag aagcctttga taagctttac aaggaagagg
     3781 acattgcaaa acttgttctt gaaattgaga accttacgaa gttagagaaa tacaagattc
     3841 gcgagttcta ccacgaaatt attggacgta agaatgacaa ggaaaacttt gcaaaaatca
     3901 tctacgaaga aatccagaat gttaataaca tgaaagagtt gatcgagaag gtaccggaca
     3961 tgagtgagtt gaaaaagagc caagtatttt acaagtatta cttagacaaa gaagagttga
     4021 acgacaagaa catcaaatac gcgttttgtc atttcgtgga aatcgccatg agtcagttgc
     4081 tgaagaacta cgtatataag cgcttaagta atatctcgaa tgacaaaatt aagcgtatct
     4141 ttgaatacca gaacttgaaa aaattgatcg aaaataagct gttaaacaaa cttgacacgt
     4201 acgtccgtaa ttgtggaaag tataattatt atttgcaaga cggcgaaatt gccacttcag
     4261 atttcatcgc ccgcaaccgt cagaatgaag cgtttcttcg caacatcatt ggggtgtcat
     4321 ctgtggccta cttttctctt cgcaacattc ttgaaacgga gaacgagaat gatattactg
     4381 ggcgtatgcg cggcaaaaca gttaagaaca ataaaggtga agagaagtac gtgtccggag
     4441 aagttgataa gatctataat gaaaataaga agaacgaggt taaggagaac ttaaaaatgt
     4501 tctattcgta cgatttcaat atggacaaca agaatgaaat cgaagatttc ttcgccaaca
     4561 tcgacgaggc gatttcttcc atccgtcacg gtattgtcca cttcaacttg gaattagaag
     4621 gtaaggatat ctttgcgttc aagaacattg cgccatccga aatctcaaag aagatgtttc
     4681 agaatgagat taacgagaaa aaactgaaat tgaagatctt tcgtcaactg aactctgcca
     4741 acgtgttccg ctatctcgaa aagtataaaa ttctgaatta ccttaaacgt acacgcttcg
     4801 agtttgtcaa taaaaatatc ccattcgtcc cgtctttcac caaattatat tcgcgcattg
     4861 atgacctgaa gaatagtctt gggatttact ggaaaactcc gaaaacaaac gacgacaata
     4921 agactaagga gattattgat gcccaaatct atttgcttaa aaacatctat tacggggagt
     4981 tcctgaatta tttcatgtcg aacaatggta atttctttga gatttctaaa gaaatcatcg
     5041 aattgaacaa gaacgataaa cgcaacttaa agactgggtt ttacaagctg caaaagtttg
     5101 aagacatcca ggagaagatt ccaaaggaat acttggcgaa tatccagtcc ctgtacatga
     5161 ttaatgccgg taatcaggac gaagaagaaa aggacactta tattgatttc attcaaaaga
     5221 tcttcttaaa gggatttatg acgtatcttg ctaataacgg tcgtttaagt ctgatttaca
     5281 tcggctcgga tgaagaaaca aatacgtcat tagcagaaaa gaagcaagag tttgacaagt
     5341 tcttgaagaa gtacgagcag aacaataata tcaagatccc ctatgagatc aatgaattcc
     5401 tgcgtgagat caaactggga aacatcctga agtatactga gcgtttaaac atgttctacc
     5461 ttatcttaaa gcttttgaat cacaaggagc tgacaaatct gaagggtagt cttgaaaaat
     5521 atcagtctgc caataaggaa gaagcgttct ctgaccaatt ggagttaatt aacctgctta
     5581 accttgacaa caaccgcgtg acggaagact tcgaattaga ggccgacgag attggaaaat
     5641 ttcttgattt caatggcaac aaagttaagg ataacaagga actgaaaaag ttcgatacaa
     5701 acaagatcta ctttgacggc gagaacatta tcaaacaccg tgccttctac aatattaaga
     5761 aatatggcat gttaaactta ctggagaaaa ttgccgacaa ggctggatac aagatctcga
     5821 tcgaagagct gaagaaatac tccaataaaa agaatgagat cgagaagaac cataagatgc
     5881 aggaaaatct gcaccgcaaa tacgctcgtc cccgtaaaga cgagaagttt acagatgagg
     5941 actatgaaag ttacaagcaa gctattgaga atattgagga gtacacccac cttaagaaca
     6001 aggtagaatt caatgagctg aatttactgc agggcctgtt gctgcgcatt ttacatcgtt
     6061 tagtcggata tacctcaatt tgggaacgcg atctgcgctt ccgccttaaa ggtgagttcc
     6121 cagaaaacca atacatcgaa gagatcttca actttgaaaa taagaagaac gtgaagtaca
     6181 aagggggtca gattgtagag aaatacatta aattctacaa ggaattacat caaaatgatg
     6241 aagttaagat caacaagtac agttccgcga atatcaaggt gttgaagcaa gaaaagaagg
     6301 acctttatat tcgcaattac atcgcccact tcaattatat tcctcacgcc gagatctcac
     6361 tgctggaagt ccttgaaaat ttgcgtaaat tgctgtccta cgatcgcaaa ctgaaaaatg
     6421 ccgtaatgaa atcagtagtt gatatcctta aggagtatgg ttttgtagcc acattcaaaa
     6481 tcggggcgga caagaagatc ggtattcaga cactggagag cgaaaaaatc gtgcatctta
     6541 agaatcttaa gaagaagaag ttaatgactg accgcaattc cgaggaactt tgcaaattgg
     6601 tgaagattat gtttgaatac aaaatggaag agaaaaagtc tgaaaactaa taacattgga
     6661 agtggataac ggatccgcga tcgcggcgcg ccacctggtg gccggccggt accacgcgtg
     6721 cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     6781 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     6841 aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga tcgcgtagtc
     6901 gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc ggtcggacag
     6961 tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta gcagcacgcc
     7021 atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg gcagtaccgg
     7081 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
     7141 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
     7201 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
     7261 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     7321 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     7381 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa cattgaaaaa
     7441 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     7501 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     7561 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     7621 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     7681 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     7741 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     7801 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     7861 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     7921 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     7981 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     8041 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     8101 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     8161 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     8221 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     8281 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     8341 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     8401 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     8461 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     8521 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     8581 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     8641 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     8701 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     8761 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     8821 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     8881 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     8941 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     9001 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     9061 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     9121 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     9181 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     9241 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     9301 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     9361 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     9421 gcgccctgac gggc
//
