LOCUS       sequence-486135-        8365 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Constitutive expression of dCas9, PspF-lambda-N22, and boxB scRNA 
            J102.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8365)
  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 8365)
  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: pCK147.02
FEATURES             Location/Qualifiers
     source          1..8365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(156..701)
                     /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(236..255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        809..843
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     protein_bind    876..894
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    931..949
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     CDS             990..1019
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1035..1052
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1108..1125)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1278..1324
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(1542..1636)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1657..2316)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2231..2250
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2317..2419)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(2623..2701)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             join(2997..3023,3024..3026,3027..5513,5514..5516,
                     5517..7103)
                     /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"
     terminator      7127..7198
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      7214..7241
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        7294..7328
                     /label=BBa_J23107
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.36"
     RBS             7338..7349
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg
       61 gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact
      121 taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac
      181 aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga
      241 taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt
      301 accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc
      361 cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg
      421 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc
      481 actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt
      541 aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt
      601 tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt
      661 tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc
      721 agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca
      781 gccccatacg atataagttg ttactagatt gacagctagc tcagtcctag gtataatact
      841 agtaggtatc cggtggaacc caagtttgag agctagggcc ctgaagaagg gccctagcaa
      901 gttcaaataa ggctagtccg ttatcaactt gggccctgaa gaagggccca agtggcaccg
      961 agtcggtgct ttttttgaag cttgggcccg aacaaaaact catctcagaa gaggatctga
     1021 atagcgccgt cgaccatcat catcatcatc attgagttta aacggtctcc agcttggctg
     1081 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     1141 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     1201 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     1261 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     1321 ttatctgttg tttgtcggtg aactggatcc ttactcgagt ctagactgca ggcttcctcg
     1381 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1441 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1501 ggccagcaaa aggccaggaa ccgtaaaaaa ctagtgcttg gattctcacc aataaaaaac
     1561 gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga
     1621 tctatcaaca ggagtccaag cgagctcgat atcaaattac gccccgccct gccactcatc
     1681 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg
     1741 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat
     1801 ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa
     1861 actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata
     1921 ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa
     1981 atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt
     2041 gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa
     2101 ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg
     2161 cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata
     2221 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat
     2281 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa
     2341 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
     2401 acctcttacg tgccgatcaa cgtctcattt tcgccagata tcgacgtctt aagttacgaa
     2461 atcatcctgt ggagcttagt aggtttagca agatggcagc gcctaaatgt agaatgataa
     2521 aaggattaag agattaattt ccctaaaaat gataaaacaa gcgttttgaa agcgcttgtt
     2581 tttttggttt gcagtcagag tagaatagaa gtatcaaaaa aagcaccgac tcggtgccac
     2641 tttttcaagt tgataacgga ctagccttat tttaacttgc tatgctgttt tgaatggttc
     2701 caacaagatt attttataac ttttataaca aataatcaag gagaaattca aagaaattta
     2761 tcagccataa aacaatactt aatactatag aatgataaca aaataaacta ctttttaaaa
     2821 gaattttgtg ttataatcta tttattatta agtattgggt aatatttttt gaagagatat
     2881 tttgaaaaag aaaaattaaa gcatattaaa ctaatttcgg aggtcattaa aactattatt
     2941 gaaatcatca aactcattat ggatttaatt taaacttttt attttaggag gcaaaaatgg
     3001 ataagaaata ctcaataggc ttagctatcg gcacaaatag cgtcggatgg gcggtgatca
     3061 ctgatgaata taaggttccg tctaaaaagt tcaaggttct gggaaataca gaccgccaca
     3121 gtatcaaaaa aaatcttata ggggctcttt tatttgacag tggagagaca gcggaagcga
     3181 ctcgtctcaa acggacagct cgtagaaggt atacacgtcg gaagaatcgt atttgttatc
     3241 tacaggagat tttttcaaat gagatggcga aagtagatga tagtttcttt catcgacttg
     3301 aagagtcttt tttggtggaa gaagacaaga agcatgaacg tcatcctatt tttggaaata
     3361 tagtagatga agttgcttat catgagaaat atccaactat ctatcatctg cgaaaaaaat
     3421 tggtagattc tactgataaa gcggatttgc gcttaatcta tttggcctta gcgcatatga
     3481 ttaagtttcg tggtcatttt ttgattgagg gagatttaaa tcctgataat agtgatgtgg
     3541 acaaactatt tatccagttg gtacaaacct acaatcaatt atttgaagaa aaccctatta
     3601 acgcaagtgg agtagatgct aaagcgattc tttctgcacg attgagtaaa tcaagacgat
     3661 tagaaaatct cattgctcag ctccccggtg agaagaaaaa tggcttattt gggaatctca
     3721 ttgctttgtc attgggtttg acccctaatt ttaaatcaaa ttttgatttg gcagaagatg
     3781 ctaaattaca gctttcaaaa gatacttacg atgatgattt agataattta ttggcgcaaa
     3841 ttggagatca atatgctgat ttgtttttgg cagctaagaa tttatcagat gctattttac
     3901 tttcagatat cctaagagta aatactgaaa taactaaggc tcccctatca gcttcaatga
     3961 ttaaacgcta cgatgaacat catcaagact tgactctttt aaaagcttta gttcgacaac
     4021 aacttccaga aaagtataaa gaaatctttt ttgatcaatc aaaaaacgga tatgcaggtt
     4081 atattgatgg gggagctagc caagaagaat tttataaatt tatcaaacca attttagaaa
     4141 aaatggatgg tactgaggaa ttattggtga aactaaatcg tgaagatttg ctgcgcaagc
     4201 aacggacctt tgacaacggc tctattcccc atcaaattca cttgggtgag ctgcatgcta
     4261 ttttgagaag acaagaagac ttttatccat ttttaaaaga caatcgtgag aagattgaaa
     4321 aaatcttgac ttttcgaatt ccttattatg ttggtccatt ggcgcgtggc aatagtcgtt
     4381 ttgcatggat gactcggaag tctgaagaaa caattacccc atggaatttt gaagaagttg
     4441 tcgataaagg tgcttcagct caatcattta ttgaacgcat gacaaacttt gataaaaatc
     4501 ttccaaatga aaaagtacta ccaaaacata gtttgcttta tgagtatttt acggtttata
     4561 acgaattgac aaaggtcaaa tatgttactg aaggaatgcg aaaaccagca tttctttcag
     4621 gtgaacagaa gaaagccatt gttgatttac tcttcaaaac aaatcgaaaa gtaaccgtta
     4681 agcaattaaa agaagattat ttcaaaaaaa tagaatgttt tgatagtgtt gaaatttcag
     4741 gagttgaaga tagatttaat gcttcattag gtacctacca tgatttgcta aaaattatta
     4801 aagataaaga ttttttggat aatgaagaaa atgaagatat cttagaggat attgttttaa
     4861 cattgacctt atttgaagat agggagatga ttgaggaaag acttaaaaca tatgctcacc
     4921 tctttgatga taaggtgatg aaacagctta aacgtcgccg ttatactggt tggggacgtt
     4981 tgtctcgaaa attgattaat ggtattaggg ataagcaatc tggcaaaaca atattagatt
     5041 ttttgaaatc agatggtttt gccaatcgca attttatgca gctgatccat gatgatagtt
     5101 tgacatttaa agaagacatt caaaaagcac aagtgtctgg acaaggcgat agtttacatg
     5161 aacatattgc aaatttagct ggtagccctg ctattaaaaa aggtatttta cagactgtaa
     5221 aagttgttga tgaattggtc aaagtaatgg ggcggcataa gccagaaaat atcgttattg
     5281 aaatggcacg tgaaaatcag acaactcaaa agggccagaa aaattcgcga gagcgtatga
     5341 aacgaatcga agaaggtatc aaagaattag gaagtcagat tcttaaagag catcctgttg
     5401 aaaatactca attgcaaaat gaaaagctct atctctatta tctccaaaat ggaagagaca
     5461 tgtatgtgga ccaagaatta gatattaatc gtttaagtga ttatgatgtc gatgccattg
     5521 ttccacaaag tttccttaaa gacgattcaa tagacaataa ggtcttaacg cgttctgata
     5581 aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt agtcaaaaag atgaaaaact
     5641 attggagaca acttctaaac gccaagttaa tcactcaacg taagtttgat aatttaacga
     5701 aagctgaacg tggaggtttg agtgaacttg ataaagctgg ttttatcaaa cgccaattgg
     5761 ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt ggatagtcgc atgaatacta
     5821 aatacgatga aaatgataaa cttattcgag aggttaaagt gattacctta aaatctaaat
     5881 tagtttctga cttccgaaaa gatttccaat tctataaagt acgtgagatt aacaattacc
     5941 atcatgccca tgatgcgtat ctaaatgccg tcgttggaac tgctttgatt aagaaatatc
     6001 caaaacttga atcggagttt gtctatggtg attataaagt ttatgatgtt cgtaaaatga
     6061 ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa atatttcttt tactctaata
     6121 tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg agagattcgc aaacgccctc
     6181 taatcgaaac taatggggaa actggagaaa ttgtctggga taaagggcga gattttgcca
     6241 cagtgcgcaa agtattgtcc atgccccaag tcaatattgt caagaaaaca gaagtacaga
     6301 caggcggatt ctccaaggag tcaattttac caaaaagaaa ttcggacaag cttattgctc
     6361 gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga tagtccaacg gtagcttatt
     6421 cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa gaagttaaaa tccgttaaag
     6481 agttactagg gatcacaatt atggaaagaa gttcctttga aaaaaatccg attgactttt
     6541 tagaagctaa aggatataag gaagttaaaa aagacttaat cattaaacta cctaaatata
     6601 gtctttttga gttagaaaac ggtcgtaaac ggatgctggc tagtgccgga gaattacaaa
     6661 aaggaaatga gctggctctg ccaagcaaat atgtgaattt tttatattta gctagtcatt
     6721 atgaaaagtt gaagggtagt ccagaagata acgaacaaaa acaattgttt gtggagcagc
     6781 ataagcatta tttagatgag attattgagc aaatcagtga attttctaag cgtgttattt
     6841 tagcagatgc caatttagat aaagttctta gtgcatataa caaacataga gacaaaccaa
     6901 tacgtgaaca agcagaaaat attattcatt tatttacgtt gacgaatctt ggagctcccg
     6961 ctgcttttaa atattttgat acaacaattg atcgtaaacg atatacgtct acaaaagaag
     7021 ttttagatgc cactcttatc catcaatcca tcactggtct ttatgaaaca cgcattgatt
     7081 tgagtcagct aggaggtgac taactcgagt aaggatctcc aggcatcaaa taaaacgaaa
     7141 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     7201 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatacct agagcgcccg
     7261 gaagagagtc aattcagggt ggtgaatctg cagtttacgg ctagctcagc cctaggtatt
     7321 atgctagcga attcattaaa gaggagaaag gtaccatggg gcccatggca gaatacaaag
     7381 ataatttact tggtgaggcg aacagctttc tcgaagtgct ggaacaggtt tcgcatctcg
     7441 caccgctgga caaaccggtg ctcatcatcg gcgaacgcgg caccggtaaa gagctgattg
     7501 ccagccgcct gcattatctc tcctcccgtt ggcaagggcc gtttatttcc cttaactgcg
     7561 cggcgttaaa tgaaaatctg ctggattccg aactgtttgg tcacgaagcg ggggcgttta
     7621 ccggtgcgca aaaacgtcat ccagggagat ttgaacgtgc cgacggcggt acgctatttc
     7681 ttgatgaact cgctacggca ccgatgatgg tgcaggagaa attattgcgc gtgattgagt
     7741 acggtgaact ggagcgcgtt ggcggcagcc aaccattgca ggtgaatgtg cggttggtat
     7801 gcgcgacgaa tgccgatctc ccggcgatgg tcaatgaagg cacttttcgc gctgacctgc
     7861 tcgaccgact ggcttttgat gttgtacaac tgccaccact gcgcgagcgc gaaagcgaca
     7921 taatgttgat ggcagaatac tttgccatcc agatgtgtcg ggaaatcaag ctgcctctgt
     7981 tcccggggtt tacggagcgc gccagagaaa cattgctgaa ttatcgttgg ccgggaaata
     8041 ttcgtgaatt gaaaaacgtg gtggaacgtt cagtgtatcg ccacggcacc agcgattatc
     8101 cgcttgatga catcattatt gatcccttta aacggcgtcc gcctgaagac gctatcgccg
     8161 tttcagaaac cacctcgctt ccaacactgc cgctggattt acgtgagttt cagatgcagc
     8221 aggaaaaaga gttgctgcaa ctcagtttgc aaatgaatgc acgcacacgc cgccgcgaac
     8281 gtcgcgcaga gaaacaggct caatggaaag cagcaaatta agcggccgcc acgcaaaaaa
     8341 ccccgcttcg gcggggtttt ttcgc
//
