LOCUS       sequence-181514-        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_K310A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     123..142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     376..393
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    408..547
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(473..492)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     633..655
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(649..840)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2298..2317
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2401..2420
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2401..2419
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2508..2530
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2550..2567
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2577..3677
                     /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     3651..3674
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3732..3752
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7364..7382)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7378..7425
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7791..7796,7797..7813,7814..7819))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7846..7864)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7932..8036
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8037..8105,8106..8897)
                     /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(8255..8274)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9068..9434,1..222)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
       61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
      421 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
      481 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
      541 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      601 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      661 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
      721 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
      781 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
      841 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
      901 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
      961 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     1021 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     1081 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     1141 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     1201 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     1261 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     1321 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     1381 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     1441 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     1501 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     1561 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     1621 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     1681 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     1741 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     1801 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1861 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1921 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1981 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     2041 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     2101 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     2161 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     2221 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     2281 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     2341 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
     2401 taatacgact cactataggg agaccacaac ggtttccctc tagtgccggc tccggagagc
     2461 tctttaatta agcggccgcc ctgcaggact cgagttctag aaataatttt gtttaacttt
     2521 aagaaggaga tatacatatg aaatcttctc accatcacca tcaccatggt tcttctatga
     2581 aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat aacggtctcg
     2641 ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt gagcatccgg
     2701 ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct gacattatct
     2761 tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct gaaatcaccc
     2821 cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta cgttacaacg
     2881 gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat aacaaagatc
     2941 tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa gaactgaaag
     3001 cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc tggccgctga
     3061 ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac attaaagacg
     3121 tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac ctgattaaaa
     3181 acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt aataaaggcg
     3241 aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc agcaaagtga
     3301 attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg ttcgttggcg
     3361 tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa gagttcctcg
     3421 aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa ccgctgggtg
     3481 ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt gccgccacta
     3541 tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc gctttctggt
     3601 atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc
     3661 tgaaagacgc gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc
     3721 tcgggatcga ggaaaacctg tacttccaat ccaatgcaat gaaagtgacg aaggtaggag
     3781 gcatttcgca taagaagtac acgtccgaag gccgcttagt gaagtcagaa tcggaagaaa
     3841 atcgcacaga cgaacgtctg tcggcgttgc ttaatatgcg ccttgacatg tatatcaaga
     3901 atcccagcag cacggaaacc aaggaaaatc aaaaacgcat tgggaaatta aagaaattct
     3961 tctcaaacaa aatggtctat cttaaagaca ataccttgag tttgaagaat gggaaaaagg
     4021 agaacattga tcgtgagtat tctgagactg acatccttga gagcgatgtc cgtgacaaga
     4081 aaaacttcgc cgtgttgaaa aagatctatc tgaatgaaaa cgtgaactcg gaggaattgg
     4141 aagtttttcg taacgacatt aagaagaaac tgaacaaaat caacagcctg aagtactcat
     4201 ttgaaaagaa taaggcgaat tatcaaaaga ttaatgagaa taacatcgag aaggttgaag
     4261 gtaagtcaaa gcgtaacatt atttacgatt attatcgtga gtcagcgaaa cgtgacgctt
     4321 atgtaagcaa tgtgaaagaa gcctttgata agctttacaa ggaagaggac attgcaaaac
     4381 ttgttcttga aattgagaac cttacgaagt tagagaaata caagattcgc gagttctacc
     4441 acgaaattat tggacgtaag aatgacaagg aaaactttgc aaaaatcatc tacgaagaaa
     4501 tccagaatgt taataacatg aaagagttga tcgagaaggt accggacatg agtgagttga
     4561 aaaagagcca agtattttac aagtattact tagacaaaga agagttgaac gacaagaaca
     4621 tcaaatacgc gttttgtcat ttcgtggaaa tcgaaatgag tcagttgctg aagaactacg
     4681 tatatgcgcg cttaagtaat atctcgaatg acaaaattaa gcgtatcttt gaataccaga
     4741 acttgaaaaa attgatcgaa aataagctgt taaacaaact tgacacgtac gtccgtaatt
     4801 gtggaaagta taattattat ttgcaagacg gcgaaattgc cacttcagat ttcatcgccc
     4861 gcaaccgtca gaatgaagcg tttcttcgca acatcattgg ggtgtcatct gtggcctact
     4921 tttctcttcg caacattctt gaaacggaga acgagaatga tattactggg cgtatgcgcg
     4981 gcaaaacagt taagaacaat aaaggtgaag agaagtacgt gtccggagaa gttgataaga
     5041 tctataatga aaataagaag aacgaggtta aggagaactt aaaaatgttc tattcgtacg
     5101 atttcaatat ggacaacaag aatgaaatcg aagatttctt cgccaacatc gacgaggcga
     5161 tttcttccat ccgtcacggt attgtccact tcaacttgga attagaaggt aaggatatct
     5221 ttgcgttcaa gaacattgcg ccatccgaaa tctcaaagaa gatgtttcag aatgagatta
     5281 acgagaaaaa actgaaattg aagatctttc gtcaactgaa ctctgccaac gtgttccgct
     5341 atctcgaaaa gtataaaatt ctgaattacc ttaaacgtac acgcttcgag tttgtcaata
     5401 aaaatatccc attcgtcccg tctttcacca aattatattc gcgcattgat gacctgaaga
     5461 atagtcttgg gatttactgg aaaactccga aaacaaacga cgacaataag actaaggaga
     5521 ttattgatgc ccaaatctat ttgcttaaaa acatctatta cggggagttc ctgaattatt
     5581 tcatgtcgaa caatggtaat ttctttgaga tttctaaaga aatcatcgaa ttgaacaaga
     5641 acgataaacg caacttaaag actgggtttt acaagctgca aaagtttgaa gacatccagg
     5701 agaagattcc aaaggaatac ttggcgaata tccagtccct gtacatgatt aatgccggta
     5761 atcaggacga agaagaaaag gacacttata ttgatttcat tcaaaagatc ttcttaaagg
     5821 gatttatgac gtatcttgct aataacggtc gtttaagtct gatttacatc ggctcggatg
     5881 aagaaacaaa tacgtcatta gcagaaaaga agcaagagtt tgacaagttc ttgaagaagt
     5941 acgagcagaa caataatatc aagatcccct atgagatcaa tgaattcctg cgtgagatca
     6001 aactgggaaa catcctgaag tatactgagc gtttaaacat gttctacctt atcttaaagc
     6061 ttttgaatca caaggagctg acaaatctga agggtagtct tgaaaaatat cagtctgcca
     6121 ataaggaaga agcgttctct gaccaattgg agttaattaa cctgcttaac cttgacaaca
     6181 accgcgtgac ggaagacttc gaattagagg ccgacgagat tggaaaattt cttgatttca
     6241 atggcaacaa agttaaggat aacaaggaac tgaaaaagtt cgatacaaac aagatctact
     6301 ttgacggcga gaacattatc aaacaccgtg ccttctacaa tattaagaaa tatggcatgt
     6361 taaacttact ggagaaaatt gccgacaagg ctggatacaa gatctcgatc gaagagctga
     6421 agaaatactc caataaaaag aatgagatcg agaagaacca taagatgcag gaaaatctgc
     6481 accgcaaata cgctcgtccc cgtaaagacg agaagtttac agatgaggac tatgaaagtt
     6541 acaagcaagc tattgagaat attgaggagt acacccacct taagaacaag gtagaattca
     6601 atgagctgaa tttactgcag ggcctgttgc tgcgcatttt acatcgttta gtcggatata
     6661 cctcaatttg ggaacgcgat ctgcgcttcc gccttaaagg tgagttccca gaaaaccaat
     6721 acatcgaaga gatcttcaac tttgaaaata agaagaacgt gaagtacaaa gggggtcaga
     6781 ttgtagagaa atacattaaa ttctacaagg aattacatca aaatgatgaa gttaagatca
     6841 acaagtacag ttccgcgaat atcaaggtgt tgaagcaaga aaagaaggac ctttatattc
     6901 gcaattacat cgcccacttc aattatattc ctcacgccga gatctcactg ctggaagtcc
     6961 ttgaaaattt gcgtaaattg ctgtcctacg atcgcaaact gaaaaatgcc gtaatgaaat
     7021 cagtagttga tatccttaag gagtatggtt ttgtagccac attcaaaatc ggggcggaca
     7081 agaagatcgg tattcagaca ctggagagcg aaaaaatcgt gcatcttaag aatcttaaga
     7141 agaagaagtt aatgactgac cgcaattccg aggaactttg caaattggtg aagattatgt
     7201 ttgaatacaa aatggaagag aaaaagtctg aaaactaata acattggaag tggataacgg
     7261 atccgcgatc gcggcgcgcc acctggtggc cggccggtac cacgcgtgcg cgctgatccg
     7321 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     7381 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     7441 atatccggat atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc
     7501 aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac
     7561 gggtgcgcat agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc
     7621 gatgctgtcg gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc
     7681 tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt
     7741 catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt
     7801 atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc
     7861 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     7921 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     7981 cgctcatgag acaataaccc tgataaatgc ttcaataaca ttgaaaaagg aagagtatga
     8041 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     8101 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     8161 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     8221 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     8281 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     8341 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     8401 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     8461 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     8521 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     8581 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     8641 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     8701 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     8761 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     8821 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     8881 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     8941 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     9001 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     9061 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     9121 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     9181 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     9241 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     9301 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     9361 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     9421 gaacgaccta cacc
//
