LOCUS       sequence-379845-        6614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 MoClo plasmid containing dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6614)
  AUTHORS   Brychcy M, Kokodynski A, Lloyd D, Godoy VG
  TITLE     AspFlex: Molecular Tools to Study Gene Expression and Regulation in 
            Acinetobacter baumannii.
  JOURNAL   ACS Synth Biol. 2023 Aug 16. doi: 10.1021/acssynbio.3c00167.
  PUBMED    37587063
REFERENCE   2  (bases 1 to 6614)
  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"; journalName: "ACS Synth 
            Biol. 2023 Aug 16. doi: 10.1021/acssynbio.3c00167."
COMMENT     Sequence Label: pBP_dCas9
FEATURES             Location/Qualifiers
     source          1..6614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(85..111,112..114,115..2601,2602..2604,2605..4191)
                     /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
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEENPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     RBS             4262..4273
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(4282..4298)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4282..4298)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4295..4317)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4306..4322
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    4341..4360
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that start with ""ATG"""
     terminator      4363..4406
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(4410..4428)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4590..5249
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4656..4675)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5270..5364
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      5547..6135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6036..6055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6289..6306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6330..6387)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
ORIGIN
        1 ggttcagctg ggtaacaccc gggttttccc agtcacgacg ttgcagaaca acagccagag
       61 agttacccgg attggtctca cataatggat aagaaatact caataggctt agctatcggc
      121 acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc
      181 aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta
      241 tttgacagtg gagagacagc ggaagcgact cgtcttaaac ggacagctcg tagaaggtat
      301 acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga gatggcgaaa
      361 gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag
      421 catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat
      481 ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc
      541 ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga
      601 gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac
      661 aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt
      721 tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag
      781 aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt
      841 aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat
      901 gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca
      961 gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata
     1021 actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg
     1081 actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt
     1141 gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt
     1201 tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa
     1261 ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat
     1321 caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt
     1381 ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt
     1441 ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca
     1501 attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt
     1561 gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc aaaacatagt
     1621 ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata tgttactgaa
     1681 ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt tgatttactc
     1741 ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt caaaaaaata
     1801 gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc ttcattaggt
     1861 acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa tgaagaaaat
     1921 gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag ggagatgatt
     1981 gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa acagcttaaa
     2041 cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg tattagggat
     2101 aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc caatcgcaat
     2161 tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca aaaagcacaa
     2221 gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg tagccctgct
     2281 attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa agtaatgggg
     2341 cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac aactcaaaag
     2401 ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga
     2461 agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga aaagctctat
     2521 ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga tattaatcgt
     2581 ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt tccttaaaga cgattcaata
     2641 gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa cgttccaagt
     2701 gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc caagttaatc
     2761 actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat
     2821 aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa gcatgtggca
     2881 caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact tattcgagag
     2941 gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga tttccaattc
     3001 tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct aaatgccgtc
     3061 gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt ctatggtgat
     3121 tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat aggcaaagca
     3181 accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga aattacactt
     3241 gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac tggagaaatt
     3301 gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat gccccaagtc
     3361 aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc aattttacca
     3421 aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa aaaatatggt
     3481 ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg
     3541 aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat ggaaagaagt
     3601 tcctttgaag aaaatccgat tgacttttta gaagctaaag gatataagga agttaaaaaa
     3661 gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg
     3721 atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc aagcaaatat
     3781 gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc agaagataac
     3841 gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat tattgagcaa
     3901 atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa agttcttagt
     3961 gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat tattcattta
     4021 tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac aacaattgat
     4081 cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca tcaatccatc
     4141 actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacta actcgaagag
     4201 accaatagaa gaagagatag cgtgcggaac ttccagcagc agtttcgggt cacccatgtc
     4261 ctttctcctc tttgtaccat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4321 aacatatgcg tctctagatt ctagaagcgg ccgcgaattc cagaaatcat ccttagcgaa
     4381 agctaaggat tttttttatc tgaaattctg cctcgtgata cgcctatttt tataggttaa
     4441 tgtcatgata ataatggttt cttagacgtc aggtggcact cgagttgatc gggcacgtaa
     4501 gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat
     4561 cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg
     4621 ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat
     4681 gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa
     4741 ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc
     4801 cggaatttcg tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt
     4861 gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg
     4921 acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc
     4981 tggcctattt ccctaaaggg tttattgaga atatgttttt cgtgtcagcc aatccctggg
     5041 tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt
     5101 tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg
     5161 ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt
     5221 actgcgatga gtggcagggc ggggcgtaat ttgatatcga gctcgcttgg actcctgttg
     5281 atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc
     5341 cgggcgtttt ttattggtga gaatccaagc ctcgagctgt cagaccaagt ttactcatat
     5401 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5461 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5521 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5581 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5641 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5701 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5761 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5821 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5881 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5941 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6001 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6061 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6121 cggagcctgt ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6181 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6241 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6301 agcgaggaag cctgcataac gcgaagtaat cttttcggtt ttaaagaaaa agggcagggt
     6361 ggtgacacct tgcccttttt tgccggactg cagctaatga gacggcatgc ggatccacta
     6421 gtgcgaaaaa accccgccct gtcaggggcg gggttttttg cgttagttgt tagacagcgg
     6481 cggcagcagc caagacagtt tacgttccgg tttggtcagc agacggatca gagaacgcag
     6541 ctgctgggac ggacggtcgg tacgagcttc ttcagagtta cgccaagaag cgaacggcgg
     6601 gtgagcagcc agac
//
