LOCUS       sequence-181385-        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_D1078A
FEATURES             Location/Qualifiers
     source          1..9434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      100..688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     589..608
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     842..859
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    874..1013
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(939..958)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1099..1121
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1115..1306)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2764..2783
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2867..2886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2867..2885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             2974..2996
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3016..3033
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3043..4143
                     /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     4117..4140
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4198..4218
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(7830..7848)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7844..7891
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(8257..8262,8263..8279,8280..8285))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(8312..8330)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8398..8502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8503..8571,8572..9363)
                     /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(8721..8740)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
       61 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
      121 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
      181 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
      241 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
      301 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
      361 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
      421 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
      481 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
      541 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
      601 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
      661 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
      721 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
      781 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
      841 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
      901 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
      961 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     1021 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     1081 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     1141 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     1201 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     1261 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     1321 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     1381 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     1441 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     1501 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     1561 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     1621 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     1681 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     1741 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     1801 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     1861 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     1921 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     1981 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     2041 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     2101 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     2161 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     2221 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     2281 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     2341 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     2401 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     2461 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     2521 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgac gctctccctt
     2581 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     2641 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     2701 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     2761 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     2821 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     2881 atagggagac cacaacggtt tccctctagt gccggctccg gagagctctt taattaagcg
     2941 gccgccctgc aggactcgag ttctagaaat aattttgttt aactttaaga aggagatata
     3001 catatgaaat cttctcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt
     3061 aaactggtaa tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag
     3121 aaattcgaga aagataccgg aattaaagtc accgttgagc atccggataa actggaagag
     3181 aaattcccac aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac
     3241 cgctttggtg gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc
     3301 caggacaagc tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct
     3361 tacccgatcg ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg
     3421 ccaaaaacct gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc
     3481 gcgctgatgt tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg
     3541 ggttatgcgt tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac
     3601 gctggcgcga aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat
     3661 gcagacaccg attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc
     3721 atcaacggcc cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg
     3781 gtactgccga ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt
     3841 attaacgccg ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg
     3901 actgatgaag gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag
     3961 tcttacgagg aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag
     4021 aaaggtgaaa tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact
     4081 gcggtgatca acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag
     4141 actaattcga gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa
     4201 aacctgtact tccaatccaa tgcaatgaaa gtgacgaagg taggaggcat ttcgcataag
     4261 aagtacacgt ccgaaggccg cttagtgaag tcagaatcgg aagaaaatcg cacagacgaa
     4321 cgtctgtcgg cgttgcttaa tatgcgcctt gacatgtata tcaagaatcc cagcagcacg
     4381 gaaaccaagg aaaatcaaaa acgcattggg aaattaaaga aattcttctc aaacaaaatg
     4441 gtctatctta aagacaatac cttgagtttg aagaatggga aaaaggagaa cattgatcgt
     4501 gagtattctg agactgacat ccttgagagc gatgtccgtg acaagaaaaa cttcgccgtg
     4561 ttgaaaaaga tctatctgaa tgaaaacgtg aactcggagg aattggaagt ttttcgtaac
     4621 gacattaaga agaaactgaa caaaatcaac agcctgaagt actcatttga aaagaataag
     4681 gcgaattatc aaaagattaa tgagaataac atcgagaagg ttgaaggtaa gtcaaagcgt
     4741 aacattattt acgattatta tcgtgagtca gcgaaacgtg acgcttatgt aagcaatgtg
     4801 aaagaagcct ttgataagct ttacaaggaa gaggacattg caaaacttgt tcttgaaatt
     4861 gagaacctta cgaagttaga gaaatacaag attcgcgagt tctaccacga aattattgga
     4921 cgtaagaatg acaaggaaaa ctttgcaaaa atcatctacg aagaaatcca gaatgttaat
     4981 aacatgaaag agttgatcga gaaggtaccg gacatgagtg agttgaaaaa gagccaagta
     5041 ttttacaagt attacttaga caaagaagag ttgaacgaca agaacatcaa atacgcgttt
     5101 tgtcatttcg tggaaatcga aatgagtcag ttgctgaaga actacgtata taagcgctta
     5161 agtaatatct cgaatgacaa aattaagcgt atctttgaat accagaactt gaaaaaattg
     5221 atcgaaaata agctgttaaa caaacttgac acgtacgtcc gtaattgtgg aaagtataat
     5281 tattatttgc aagacggcga aattgccact tcagatttca tcgcccgcaa ccgtcagaat
     5341 gaagcgtttc ttcgcaacat cattggggtg tcatctgtgg cctacttttc tcttcgcaac
     5401 attcttgaaa cggagaacga gaatgatatt actgggcgta tgcgcggcaa aacagttaag
     5461 aacaataaag gtgaagagaa gtacgtgtcc ggagaagttg ataagatcta taatgaaaat
     5521 aagaagaacg aggttaagga gaacttaaaa atgttctatt cgtacgattt caatatggac
     5581 aacaagaatg aaatcgaaga tttcttcgcc aacatcgacg aggcgatttc ttccatccgt
     5641 cacggtattg tccacttcaa cttggaatta gaaggtaagg atatctttgc gttcaagaac
     5701 attgcgccat ccgaaatctc aaagaagatg tttcagaatg agattaacga gaaaaaactg
     5761 aaattgaaga tctttcgtca actgaactct gccaacgtgt tccgctatct cgaaaagtat
     5821 aaaattctga attaccttaa acgtacacgc ttcgagtttg tcaataaaaa tatcccattc
     5881 gtcccgtctt tcaccaaatt atattcgcgc attgatgacc tgaagaatag tcttgggatt
     5941 tactggaaaa ctccgaaaac aaacgacgac aataagacta aggagattat tgatgcccaa
     6001 atctatttgc ttaaaaacat ctattacggg gagttcctga attatttcat gtcgaacaat
     6061 ggtaatttct ttgagatttc taaagaaatc atcgaattga acaagaacga taaacgcaac
     6121 ttaaagactg ggttttacaa gctgcaaaag tttgaagaca tccaggagaa gattccaaag
     6181 gaatacttgg cgaatatcca gtccctgtac atgattaatg ccggtaatca ggacgaagaa
     6241 gaaaaggaca cttatattga tttcattcaa aagatcttct taaagggatt tatgacgtat
     6301 cttgctaata acggtcgttt aagtctgatt tacatcggct cggatgaaga aacaaatacg
     6361 tcattagcag aaaagaagca agagtttgac aagttcttga agaagtacga gcagaacaat
     6421 aatatcaaga tcccctatga gatcaatgaa ttcctgcgtg agatcaaact gggaaacatc
     6481 ctgaagtata ctgagcgttt aaacatgttc taccttatct taaagctttt gaatcacaag
     6541 gagctgacaa atctgaaggg tagtcttgaa aaatatcagt ctgccaataa ggaagaagcg
     6601 ttctctgacc aattggagtt aattaacctg cttaaccttg acaacaaccg cgtgacggaa
     6661 gacttcgaat tagaggccga cgagattgga aaatttcttg atttcaatgg caacaaagtt
     6721 aaggataaca aggaactgaa aaagttcgat acaaacaaga tctactttga cggcgagaac
     6781 attatcaaac accgtgcctt ctacaatatt aagaaatatg gcatgttaaa cttactggag
     6841 aaaattgccg acaaggctgg atacaagatc tcgatcgaag agctgaagaa atactccaat
     6901 aaaaagaatg agatcgagaa gaaccataag atgcaggaaa atctgcaccg caaatacgct
     6961 cgtccccgta aagacgagaa gtttacagat gaggactatg aaagttacaa gcaagctatt
     7021 gagaatattg aggagtacac ccaccttaag aacaaggtag aattcaatga gctgaattta
     7081 ctgcagggcc tgttgctgcg cattttacat cgtttagtcg gatatacctc aatttgggaa
     7141 cgcgatctgc gcttccgcct taaaggtgag ttcccagaaa accaatacat cgaagagatc
     7201 ttcaactttg aaaataagaa gaacgtgaag tacaaagggg gtcagattgt agagaaatac
     7261 attaaattct acaaggaatt acatcaaaat gatgaagtta agatcaacaa gtacagttcc
     7321 gcgaatatca aggtgttgaa gcaagaaaag aaggaccttt atattcgcaa ttacatcgcc
     7381 cacttcaatt atattcctca cgccgagatc tcactgctgg aagtccttga aaatttgcgt
     7441 aaattgctgt cctacgcacg caaactgaaa aatgccgtaa tgaaatcagt agttgatatc
     7501 cttaaggagt atggttttgt agccacattc aaaatcgggg cggacaagaa gatcggtatt
     7561 cagacactgg agagcgaaaa aatcgtgcat cttaagaatc ttaagaagaa gaagttaatg
     7621 actgaccgca attccgagga actttgcaaa ttggtgaaga ttatgtttga atacaaaatg
     7681 gaagagaaaa agtctgaaaa ctaataacat tggaagtgga taacggatcc gcgatcgcgg
     7741 cgcgccacct ggtggccggc cggtaccacg cgtgcgcgct gatccggctg ctaacaaagc
     7801 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     7861 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc
     7921 acaggacggg tgtggtcgcc atgatcgcgt agtcgatagt ggctccaagt agcgaagcga
     7981 gcaggactgg gcggcggcca aagcggtcgg acagtgctcc gagaacgggt gcgcatagaa
     8041 attgcatcaa cgcatatagc gctagcagca cgccatagtg actggcgatg ctgtcggaat
     8101 ggacgatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
     8161 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
     8221 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct
     8281 gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     8341 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     8401 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     8461 taaccctgat aaatgcttca ataacattga aaaaggaaga gtatgagtat tcaacatttc
     8521 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     8581 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     8641 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     8701 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     8761 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     8821 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     8881 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     8941 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     9001 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     9061 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     9121 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     9181 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     9241 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     9301 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     9361 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     9421 tttaaaagga tcta
//
