LOCUS       sequence-486137-        8310 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Constitutive expression of dCas9, MCP-SYNZIP6, and 
            alpha-NTD-SYNZIP5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8310)
  AUTHORS   Kiattisewee C, Karanjia AV, Cardiff RAL, Olander KE, Leejareon P, 
            Alvi SS, Carothers JM, Zalatan JG
  TITLE     Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene
            Activation Reveals Antagonistic Effects.
  JOURNAL   ACS Synth Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358.
  PUBMED    40693287
REFERENCE   2  (bases 1 to 8310)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jul 22. doi: 10.1021/acssynbio.5c00358."
COMMENT     Sequence Label: pCK157
FEATURES             Location/Qualifiers
     source          1..8310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      347..418
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      434..461
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        518..552
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             562..573
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             2196..2207
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(2217..2251)
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     rep_origin      complement(2413..2958)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2493..2512)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3072..3166)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3187..3846)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3761..3780
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3847..3949)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(4153..4231)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(4527..4553,4554..4556,4557..7043,7044..7046,7047..8310,
                     1..323)
                     /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
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 ataagcatta tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt
       61 tagcagatgc caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa
      121 tacgtgaaca agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg
      181 ctgcttttaa atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag
      241 ttttagatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
      301 tgagtcagct aggaggtgac taactcgagt aaggatctcc aggcatcaaa taaaacgaaa
      361 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
      421 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatacct agagcgcccg
      481 gaagagagtc aattcagggt ggtgaatccc cagatctttt acggctagct cagccctagg
      541 tattatgcta gcgaattcat taaagaggag aaaggtacca tggggcccgc ttctaacttt
      601 actcagttcg ttctcgtcga caatggcgga actggcgacg tgactgtcgc cccaagcaac
      661 ttcgctaacg ggatcgctga atggatcagc tctaactcgc gttcacaggc ttacaaagta
      721 acctgtagcg ttcgtcagag ctctgcgcag aatcgcaaat acaccatcaa agtcgaggtg
      781 cctaaaggcg cctggcgttc gtacttaaat atggaactaa ccattccaat tttcgccacg
      841 aattccgact gcgagcttat tgttaaggca atgcaaggtc tcctaaaaga tggaaacccg
      901 attccctcag caatcgcagc aaactccggc atctacggtg gcggaggtag ccaaaaagtt
      961 gcgcagctga aaaaccgtgt tgcgtacaaa ctgaaagaaa acgcgaagct ggagaacatc
     1021 gtggcgcgtc tggaaaacga caatgcgaac ctggagaaag acattgcgaa tctcgaaaag
     1081 gacatcgcaa atctggaacg tgacgttgcg cgttaagcgg ccgccacgca aaaaaccccg
     1141 cttcggcggg gttttttcgc cctagggata tattccgctt cctcgctcac tgactcgcta
     1201 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg
     1261 cccggggcga aaaaaccccg ccgaagcggg gttttttgcg tgggcgcgcc ttactcgaat
     1321 ttgtgagccg ccagttcgaa ttccagttcc gcttttgcga atttcaggtg ttccttcagg
     1381 tttttgagtt cagcgttacg ctcttccagc tcctggatgt agtttttcag ttctttaacg
     1441 gtgttctctg gtttctcttc tttcacttca ggctgacgta catcacgtaa gtcaacgaaa
     1501 gcttccagtt gttcagccag aatggttgcc gcacgacgaa tcgcctcttc aggatcgatt
     1561 gtgccgttgg tttccatttc gatgaccagc ttgtccaggt cggtacgctg ttctacacgc
     1621 gctgcttcaa cattgtaggc aatacgctcc acagggctgt agcatgcgtc gaccagcaga
     1681 cggccgattg ggcgctcatc ttcttccgaa tgaattcggg tagaagccgg cacataacca
     1741 cgaccgcgct gaactttgat acgcatgcta atagacgcgt tctcatcggt caggtggcag
     1801 atcacgtgct gcggcttgac gatttcgaca tcaccgtcgt gggtgatatc ggctgcagtc
     1861 acagggccaa tgccagattt attcaaggta agaataactt catctttgcc ctgaactctc
     1921 accgccagcc ctttcaggtt gagcaggatt tccaggatat cttcctgaac gccttctttg
     1981 gtgctgtact catgtagtac accatcaatc tcaacctcgg tcaccgcgca acccggcatc
     2041 gatgagagca gaatacggcg cagtgcgtta cccagagtat ggccaaagcc acgctctaaa
     2101 ggctcaaggg tcaccttggc gtgcgtcgaa ctcacttgct cgatatcaac caggcgcggt
     2161 tttagaaact ctgtcacaga accctgcatg gtacctttct cctctttaat gaattcgcta
     2221 gcataatacc tagggctgag ctagccgtaa aatgcatcct agggatatat tccgcttcct
     2281 cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg gcttacgaac
     2341 ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg agagggccgc
     2401 ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc
     2461 aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggcgg
     2521 ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc cgctgttatg
     2581 gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc
     2641 tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc
     2701 gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt
     2761 gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga aaggacaagt
     2821 tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt agctcagaga
     2881 accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc
     2941 agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt ctagatttca
     3001 gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtta
     3061 ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa
     3121 tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctcgat
     3181 atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
     3241 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     3301 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
     3361 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     3421 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     3481 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     3541 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     3601 cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag
     3661 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     3721 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     3781 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     3841 ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
     3901 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt
     3961 tcgccagata tcgacgtctt aagttacgaa atcatcctgt ggagcttagt aggtttagca
     4021 agatggcagc gcctaaatgt agaatgataa aaggattaag agattaattt ccctaaaaat
     4081 gataaaacaa gcgttttgaa agcgcttgtt tttttggttt gcagtcagag tagaatagaa
     4141 gtatcaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     4201 tttaacttgc tatgctgttt tgaatggttc caacaagatt attttataac ttttataaca
     4261 aataatcaag gagaaattca aagaaattta tcagccataa aacaatactt aatactatag
     4321 aatgataaca aaataaacta ctttttaaaa gaattttgtg ttataatcta tttattatta
     4381 agtattgggt aatatttttt gaagagatat tttgaaaaag aaaaattaaa gcatattaaa
     4441 ctaatttcgg aggtcattaa aactattatt gaaatcatca aactcattat ggatttaatt
     4501 taaacttttt attttaggag gcaaaaatgg ataagaaata ctcaataggc ttagctatcg
     4561 gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt
     4621 tcaaggttct gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt
     4681 tatttgacag tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt
     4741 atacacgtcg gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga
     4801 aagtagatga tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga
     4861 agcatgaacg tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat
     4921 atccaactat ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc
     4981 gcttaatcta tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg
     5041 gagatttaaa tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct
     5101 acaatcaatt atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc
     5161 tttctgcacg attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg
     5221 agaagaaaaa tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt
     5281 ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg
     5341 atgatgattt agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg
     5401 cagctaagaa tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa
     5461 taactaaggc tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact
     5521 tgactctttt aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt
     5581 ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat
     5641 tttataaatt tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga
     5701 aactaaatcg tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc
     5761 atcaaattca cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat
     5821 ttttaaaaga caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg
     5881 ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa
     5941 caattacccc atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta
     6001 ttgaacgcat gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata
     6061 gtttgcttta tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg
     6121 aaggaatgcg aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac
     6181 tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa
     6241 tagaatgttt tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag
     6301 gtacctacca tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa
     6361 atgaagatat cttagaggat attgttttaa cattgacctt atttgaagat agggagatga
     6421 ttgaggaaag acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta
     6481 aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg
     6541 ataagcaatc tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca
     6601 attttatgca gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac
     6661 aagtgtctgg acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg
     6721 ctattaaaaa aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg
     6781 ggcggcataa gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa
     6841 agggccagaa aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag
     6901 gaagtcagat tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct
     6961 atctctatta tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc
     7021 gtttaagtga ttatgatgtc gatgccattg ttccacaaag tttccttaaa gacgattcaa
     7081 tagacaataa ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa
     7141 gtgaagaagt agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa
     7201 tcactcaacg taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg
     7261 ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg
     7321 cacaaatttt ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag
     7381 aggttaaagt gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat
     7441 tctataaagt acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg
     7501 tcgttggaac tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg
     7561 attataaagt ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag
     7621 caaccgcaaa atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac
     7681 ttgcaaatgg agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa
     7741 ttgtctggga taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag
     7801 tcaatattgt caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac
     7861 caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg
     7921 gtggttttga tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag
     7981 ggaaatcgaa gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa
     8041 gttcctttga aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa
     8101 aagacttaat cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac
     8161 ggatgctggc tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat
     8221 atgtgaattt tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata
     8281 acgaacaaaa acaattgttt gtggagcagc
//
