LOCUS       sequence-233418-        6875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled plasmid - Encodes dCas9 with no targeting sgRNA (used 
            in Stage 2 assembly to repress target genes).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6875)
  AUTHORS   Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar 
            S, Davies BW, Ellington AD, Moran NA, Barrick JE
  TITLE     Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit 
            for Modular Assembly of Broad-Host-Range Plasmids.
  JOURNAL   ACS Synth Biol. 2018 May 18;7(5):1279-1290. doi: 
            10.1021/acssynbio.7b00399. Epub 2018 Apr 13.
  PUBMED    29608282
REFERENCE   2  (bases 1 to 6875)
  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: 
            "10.1021/acssynbio.7b00399"; journalName: "ACS Synth Biol"; date: 
            "2018-05-18- 18"; volume: "7"; issue: "5"; pages: "1279-1290"
COMMENT     Sequence Label: pBTK614
FEATURES             Location/Qualifiers
     source          1..6875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(366..1157,1158..1226))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     989..1008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1227..1331)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1434..2022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1923..1942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(2472..2498,2499..2501,2502..4988,4989..4991,
                     4992..6578)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRHKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     misc_feature    6599..6730
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   6731..6766
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     terminator      6782..6830
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
ORIGIN
        1 ctgccagaca tcaaataaaa caaaaggctc agtcggaaga ctgggccttt tgttttatct
       61 gttgtttgtc ggtgaacact ctcccggctg agcatgagac ggaaatctgc tcgtcagtgg
      121 tgctcacact gacgaatcat gtacagatca taccgatgac tgcctggcga ctcacaacta
      181 agcaagacag ccggaaccag cgccggcgaa caccactgca tatatggcat atcacaacag
      241 tccaactagt gcactgcagt acagcggccg cgattatcaa aaaggatctt cacctagatc
      301 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
      361 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
      421 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
      481 ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga tttatcagca
      541 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
      601 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
      661 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
      721 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
      781 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
      841 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
      901 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
      961 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     1021 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     1081 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     1141 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     1201 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     1261 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     1321 ggggttccgc gcacatttcc ccgaaaagtg ccacctgtca tgaccaaaat cccttaacgt
     1381 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1441 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1501 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1561 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     1621 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1681 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1741 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1801 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1861 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1921 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     1981 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2041 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2101 cctgattctg tggataaccg tgcggccgcc cctgaattcg catctagaac ggggtcatca
     2161 cggctcatca tgcgccaaac aaatgtgtgc aatacacgct cggatgactg catgatgacc
     2221 gcactgactg gggacagcag atccacctaa gcctgtgaga gaagcagaca cccgacagat
     2281 caaggcagtt acgtctcacc aaaccagatg tcaacacagc tacctgatcc ttcaactcag
     2341 caaaagttcg atttattcaa caaagccacg ttgtgtctca aaatctctga tgttacattg
     2401 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     2461 caaggggtgt tatggataag aaatactcaa taggcttagc tatcggcaca aatagcgtcg
     2521 gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag gttctgggaa
     2581 atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt gacagtggag
     2641 agacagcgga agcgactcgt cataaacgga cagctcgtag aaggtataca cgtcggaaga
     2701 atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta gatgatagtt
     2761 tctttcatcg acttgaagag tcttttttgg tggaagaaga caagaagcat gaacgtcatc
     2821 ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca actatctatc
     2881 atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta atctatttgg
     2941 ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat ttaaatcctg
     3001 ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat caattatttg
     3061 aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct gcacgattga
     3121 gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag aaaaatggct
     3181 tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa tcaaattttg
     3241 atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat gatttagata
     3301 atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct aagaatttat
     3361 cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact aaggctcccc
     3421 tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact cttttaaaag
     3481 ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat caatcaaaaa
     3541 acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat aaatttatca
     3601 aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta aatcgtgaag
     3661 atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa attcacttgg
     3721 gtgagctgca tgctattttg agaagacaag aagactttta tccattttta aaagacaatc
     3781 gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt ccattggcgc
     3841 gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt accccatgga
     3901 attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa cgcatgacaa
     3961 actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg ctttatgagt
     4021 attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga atgcgaaaac
     4081 cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc aaaacaaatc
     4141 gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa tgttttgata
     4201 gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc taccatgatt
     4261 tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa gatatcttag
     4321 aggatattgt tttaacattg accttatttg aagataggga gatgattgag gaaagactta
     4381 aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt cgccgttata
     4441 ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag caatctggca
     4501 aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt atgcagctga
     4561 tccatgatga tagtttgaca tttaaagaag acattcaaaa agcacaagtg tctggacaag
     4621 gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt aaaaaaggta
     4681 ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg cataagccag
     4741 aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc cagaaaaatt
     4801 cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt cagattctta
     4861 aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc tattatctcc
     4921 aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta agtgattatg
     4981 atgtcgatgc cattgttcca caaagtttcc ttaaagacga ttcaatagac aataaggtct
     5041 taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa gaagtagtca
     5101 aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact caacgtaagt
     5161 ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa gctggtttta
     5221 tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa attttggata
     5281 gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt aaagtgatta
     5341 ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat aaagtacgtg
     5401 agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt ggaactgctt
     5461 tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat aaagtttatg
     5521 atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc gcaaaatatt
     5581 tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca aatggagaga
     5641 ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc tgggataaag
     5701 ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat attgtcaaga
     5761 aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa agaaattcgg
     5821 acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt tttgatagtc
     5881 caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa tcgaagaagt
     5941 taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc tttgaaaaaa
     6001 atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac ttaatcatta
     6061 aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg ctggctagtg
     6121 ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg aattttttat
     6181 atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa caaaaacaat
     6241 tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc agtgaatttt
     6301 ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca tataacaaac
     6361 atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt acgttgacga
     6421 atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt aaacgatata
     6481 cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact ggtctttatg
     6541 aaacacgcat tgatttgagt cagctaggag gtgactgaag tatattttag atgaagatta
     6601 tttcttaata actaaaaata tggtataata ctcttaataa atgcagtaat acaggggctt
     6661 ttcaagactg aagtctagct gagacaaata gtgcgattac gaaatttttt agacaaaaat
     6721 agtctacgag gttttagagc tatgctgttt tgaatggtcc caaaacaaag caagctgata
     6781 aaccgataca attaaaggct ccttttggag cctttttttt tggagatttt caacatgaag
     6841 aaattattat ttgatgatca gatagcggcg gggaa
//
