LOCUS       sequence-75536-e        7090 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CcaS and CcaR constitutively, and sfGFP/TetR under the 
            PcpcG2 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7090)
  AUTHORS   Olson EJ, Hartsough LA, Landry BP, Shroff R, Tabor JJ
  TITLE     Characterizing bacterial gene circuit dynamics with optically 
            programmed gene expression signals.
  JOURNAL   Nat Methods. 2014 Mar 9. doi: 10.1038/nmeth.2884.
  PUBMED    24608181
REFERENCE   2  (bases 1 to 7090)
  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: "Nat 
            Methods. 2014 Mar 9. doi: 10.1038/nmeth.2884."
COMMENT     Sequence Label: pBL4
FEATURES             Location/Qualifiers
     source          1..7090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2947..3049
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             3050..3709
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3116..3135)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3730..3824
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      3912..4500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4401..4420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4665..4751)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             5706..5717
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             5724..6437
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             6464..7090
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(6540..6559)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 tttttttgac ggtaaagcca agctgaaggc tgacaaaatc tatactgtta tatccccctt
       61 tcctggggga cttaggcggt ttaataactg gggtttttgc tagaaatatt ttggcgttta
      121 attatttatt ctctggttta atatttcgag actttgcaga ctttgtattt gaggccagac
      181 tccaatgttg aaactagccg gtaaaaaagc ctctgccctg acctgcaaga aaacgaaggc
      241 aaaaattggt atcctagaaa tggatcggca gtggtatcaa tcagtcaagc gatttttatg
      301 tccaagtttt gaaatttctg ctatcaactt ccaagatatt ttgtttaaca atgttgacgt
      361 tggagaatat cattcgatcc tagttggttg cgggattaaa tatgaatcaa ttgatatttc
      421 agctacttta gatttagtga ccattattaa aaaattgtct gctaagccgc cggccctact
      481 actaattacg gattgtgccg acagtcagat cacggtacaa gctcgcagtt acctgcccca
      541 aattgacggc gtttttgcca aggatcatga tttagcttta ttgttaaaag tcatgaaaat
      601 tattgccaaa caaaaatatt ttaagtaagg tttttctact gggattcgct aatatatggg
      661 caaatttcta attccaatcg aatttgtttt tctggcgatc gccatgacct gttatttatg
      721 gcacagacaa aaccaagaac gccgcaggat tgaaattagc atcaagcaac aaacccaacg
      781 ggaacgattt attaaccaaa ttacccaaca tatccgccaa tctttaaact tggaaacggt
      841 tttaaatacc accgtcgctg aagttaaaac cctgttgcaa gttgatcgag ttctaattta
      901 tcgcatttgg caagatggca cgggcagcgc cattacggaa tcggtgaatg ccaattatcc
      961 tagtatttta gggcggacct tttccgatga agtttttccc gttgaatacc atcaagccta
     1021 caccaaaggt aaagtacggg ccattaatga cattgaccag gatgacatag agatttgcct
     1081 agctgatttc gtcaaacaat ttggcgtgaa atcaaaatta gtagtgccca ttcttcaaca
     1141 taatcgtgct tcttccctag ataatgaatc agaatttccc tatctttggg ggctgttaat
     1201 tacccatcaa tgtgctttta cccggccatg gcaaccgtgg gaagtggagt taatgaaaca
     1261 gctagccaat caggtcgcga tcgccatcca acaatcggaa ttatatgagc aattacagca
     1321 actcaataaa gatttggaaa accgagtcga aaaacgcacc cagcaacttg ccgccaccaa
     1381 tcaatcccta agaatggaaa tcagtgagcg acaaaaaacg gaagccgctc tccgccacac
     1441 taaccatact ctgcaatccc tgattgcggc ctcccccagg ggtattttta cccttaattt
     1501 agcagaccaa attcagattt ggaatcctac agcagaacgt atttttggtt ggacagaaac
     1561 agaaattatt gcccatccag aattattaac atccaacatt ttgctggaag attatcagca
     1621 atttaaacag aaagttttat caggcatggt ttcccctagc ctagaattaa aatgtcaaaa
     1681 aaaagatggt agttggattg aaattgtcct ttccgctgct cccctattgg atagtgaaga
     1741 aaatattgcc ggattggtgg cggttgtcgc cgatattacc gagcaaaagc ggcaggcaga
     1801 acaaattcgt ttgctacaat ccgttgtggt taatactaat gatgcggtgg tgattacgga
     1861 agcggagccc attgatgatc ccgggccgag aattctctat gtcaatgaag catttactaa
     1921 aatcaccggt tatactgctg aagaaatgct aggcaaaacc ccccgagttt tacagggacc
     1981 aaaaactagt cgcactgaat tagatagggt gcggcaagcc attagtcaat ggcaatcagt
     2041 taccgttgaa gtgattaatt atcgtaagga tggcagtgag ttttgggtgg aatttagtct
     2101 ggtgcccgtt gccaataaaa caggttttta cacccattgg attgctgtgc aaagggatgt
     2161 cactgagcgc cgacgcacgg aggaagtccg cctagcttta gaacgggaaa aagaattaag
     2221 ccgcctaaaa actcgttttt tctccatggc ttcccatgaa tttcgtactc ccctcagtac
     2281 ggccttagct gctgcccaat tactggaaaa ttctgaagtg gcctggcttg atcccgataa
     2341 gcgtagccgg aacttacacc gtattcaaaa ttccgtgaaa aatatggtac agctcctgga
     2401 tgatatttta atcattaacc gtgccgaagc gggcaaattg gaatttaatc ctaattggtt
     2461 agatttgaaa ttattgttcc agcaatttat cgaagaaatt caattaagtg tcagtgacca
     2521 atattatttt gactttattt gtagcgctca agatacgaag gcattggtgg atgaaaggtt
     2581 agtgcggtct attttatcta atctgttatc taatgcgatt aaatactctc ccgggggagg
     2641 gcagattaaa attgccctaa gcctagattc ggaacagatt atttttgaag tcaccgacca
     2701 gggcattggc atttcgccag aggaccaaaa gcaaattttt gaaccctttc atcggggcaa
     2761 aaatgtcaga aatattacgg gaacaggact cggtttaatg gttgccaaga aatgtgttga
     2821 cttacacagt ggcagtatct tgctaaaaag tgcagttgac cagggaacaa cagttactat
     2881 ctgtttaaaa cgctataacc atttgcctcg agcttaggac gtcgatatct ggcgaaaatg
     2941 agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc
     3001 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     3061 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     3121 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     3181 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     3241 ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg agctggtgat
     3301 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     3361 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     3421 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     3481 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     3541 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     3601 gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat
     3661 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttgatatcga
     3721 gctcgcttgg actcctgttg atagatccag taatgacctc agaactccat ctggatttgt
     3781 tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc actagtcatg
     3841 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3901 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3961 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4021 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4081 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4141 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4201 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4261 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
     4321 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4381 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4441 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4501 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4561 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4621 gataccgctc gccgcagccg aacgccctag gtctagggcg gcggatttgt cctactcagg
     4681 agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt
     4741 cgttttattt gatgcctcta gactagtttt tcccttggca caaagatttt tccgttaaat
     4801 tggccagacg atacccaatg ccatggaccg tttcaatggc atctgctgat aaaccggcac
     4861 tttttaattt ttgtcgcaaa ctccgcacat gcaccttaac cgtatcttcc tctgggggac
     4921 tctccaactt ccagatacta tcgataatca tgctccgact tagcacccga cggccattgc
     4981 ggagtagtaa ttccagaatg ctgtattcct tgcgggtcaa agacaaaacc tcattgtcat
     5041 aactaacttc ataggtgctt ggatccaacc tgattggccc ccactctaaa actggttggc
     5101 acgttgcaca accccgacgc aacaaagctc gcaccctggc aaataactcc cccaaatcca
     5161 ctggcttgac cacataatca tccgcccccg catccaagcc cgtgatttta tcattgatcg
     5221 tatccctggc tgtcatcatc aaaattggca ttaaataact gtgcgatcgc catttttgac
     5281 agagggtaat cccgtccaac tccggcagca tcacatccaa aataacgagg tcatattcca
     5341 gtcgggaggc atagtcccag gcgagggaag cgtcggtggc aatatcaacg gtgtaaagct
     5401 ggtcactcaa tgcttcagca agggtttccg ccagcggcaa atcatcctcc actaaaagaa
     5461 ttctcatagc ccattgtgct tttctctatc aacctcagct tacctgaagg ggtgaacagg
     5521 tctgggttaa ttcatgttgc gaaatgtaac agttttagtc gcatcagcta actttccgat
     5581 ttctttacga ttttctcccc cttttcttca attttacttt gttaggatcg catttttaat
     5641 gccaacacat accagttatt ggctggacat taaacaactt ttaagtttaa ttactaactt
     5701 tatctaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc
     5761 ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg cgtggcgagg
     5821 gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact actggtaaac
     5881 tgccggtacc ttggccgact ctggtaacga cgctgactta tggtgttcag tgctttgctc
     5941 gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg gaaggctatg
     6001 tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt gcggaagtga
     6061 aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac tttaaagaag
     6121 acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat gtttacatca
     6181 ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac aacgtggagg
     6241 atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt gatggtcctg
     6301 ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg
     6361 agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc acgcatggta
     6421 tggatgaact gtacaaatga tgatcacaca ggaaacctac tagatgatgt ctcgtttaga
     6481 taaaagtaaa gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt
     6541 aacaacccgt aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt
     6601 aaaaaataag cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac
     6661 tcacttttgc cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag
     6721 ttttagatgt gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc
     6781 tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt
     6841 ttcactagag aatgcattat atgcactcag cgcagtgggg cattttactt taggttgcgt
     6901 attggaagat caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag
     6961 tatgccgcca ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc
     7021 cttcttattc ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag
     7081 tgggtcttaa
//
