LOCUS       sequence-476782-        7090 bp    DNA     circular SYN 20-JUN-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 Jun 20, 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"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(754..840)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             1795..1806
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1813..2526
                     /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             2553..3179
                     /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(2629..2648)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6126..6228
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             6229..6888
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(6295..6314)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      6909..7003
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgccctagg tctagggcgg cggatttgtc ctactcagga gagcgttcac
      781 cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg
      841 atgcctctag actagttttt cccttggcac aaagattttt ccgttaaatt ggccagacga
      901 tacccaatgc catggaccgt ttcaatggca tctgctgata aaccggcact ttttaatttt
      961 tgtcgcaaac tccgcacatg caccttaacc gtatcttcct ctgggggact ctccaacttc
     1021 cagatactat cgataatcat gctccgactt agcacccgac ggccattgcg gagtagtaat
     1081 tccagaatgc tgtattcctt gcgggtcaaa gacaaaacct cattgtcata actaacttca
     1141 taggtgcttg gatccaacct gattggcccc cactctaaaa ctggttggca cgttgcacaa
     1201 ccccgacgca acaaagctcg caccctggca aataactccc ccaaatccac tggcttgacc
     1261 acataatcat ccgcccccgc atccaagccc gtgattttat cattgatcgt atccctggct
     1321 gtcatcatca aaattggcat taaataactg tgcgatcgcc atttttgaca gagggtaatc
     1381 ccgtccaact ccggcagcat cacatccaaa ataacgaggt catattccag tcgggaggca
     1441 tagtcccagg cgagggaagc gtcggtggca atatcaacgg tgtaaagctg gtcactcaat
     1501 gcttcagcaa gggtttccgc cagcggcaaa tcatcctcca ctaaaagaat tctcatagcc
     1561 cattgtgctt ttctctatca acctcagctt acctgaaggg gtgaacaggt ctgggttaat
     1621 tcatgttgcg aaatgtaaca gttttagtcg catcagctaa ctttccgatt tctttacgat
     1681 tttctccccc ttttcttcaa ttttactttg ttaggatcgc atttttaatg ccaacacata
     1741 ccagttattg gctggacatt aaacaacttt taagtttaat tactaacttt atctaaagag
     1801 gagaaatact agatgcgtaa aggcgaagag ctgttcactg gtgtcgtccc tattctggtg
     1861 gaactggatg gtgatgtcaa cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac
     1921 gcaactaatg gtaaactgac gctgaagttc atctgtacta ctggtaaact gccggtacct
     1981 tggccgactc tggtaacgac gctgacttat ggtgttcagt gctttgctcg ttatccggac
     2041 catatgaagc agcatgactt cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc
     2101 acgatttcct ttaaggatga cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc
     2161 gataccctgg taaaccgcat tgagctgaaa ggcattgact ttaaagaaga cggcaatatc
     2221 ctgggccata agctggaata caattttaac agccacaatg tttacatcac cgccgataaa
     2281 caaaaaaatg gcattaaagc gaattttaaa attcgccaca acgtggagga tggcagcgtg
     2341 cagctggctg atcactacca gcaaaacact ccaatcggtg atggtcctgt tctgctgcca
     2401 gacaatcact atctgagcac gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat
     2461 catatggttc tgctggagtt cgtaaccgca gcgggcatca cgcatggtat ggatgaactg
     2521 tacaaatgat gatcacacag gaaacctact agatgatgtc tcgtttagat aaaagtaaag
     2581 tgattaacag cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta
     2641 aactcgccca gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc
     2701 gggctttgct cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc
     2761 ctttagaagg ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg
     2821 ctttactaag tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac
     2881 agtatgaaac tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga
     2941 atgcattata tgcactcagc gcagtggggc attttacttt aggttgcgta ttggaagatc
     3001 aagagcatca agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat
     3061 tattacgaca agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg
     3121 gccttgaatt gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtcttaat
     3181 ttttttgacg gtaaagccaa gctgaaggct gacaaaatct atactgttat atcccccttt
     3241 cctgggggac ttaggcggtt taataactgg ggtttttgct agaaatattt tggcgtttaa
     3301 ttatttattc tctggtttaa tatttcgaga ctttgcagac tttgtatttg aggccagact
     3361 ccaatgttga aactagccgg taaaaaagcc tctgccctga cctgcaagaa aacgaaggca
     3421 aaaattggta tcctagaaat ggatcggcag tggtatcaat cagtcaagcg atttttatgt
     3481 ccaagttttg aaatttctgc tatcaacttc caagatattt tgtttaacaa tgttgacgtt
     3541 ggagaatatc attcgatcct agttggttgc gggattaaat atgaatcaat tgatatttca
     3601 gctactttag atttagtgac cattattaaa aaattgtctg ctaagccgcc ggccctacta
     3661 ctaattacgg attgtgccga cagtcagatc acggtacaag ctcgcagtta cctgccccaa
     3721 attgacggcg tttttgccaa ggatcatgat ttagctttat tgttaaaagt catgaaaatt
     3781 attgccaaac aaaaatattt taagtaaggt ttttctactg ggattcgcta atatatgggc
     3841 aaatttctaa ttccaatcga atttgttttt ctggcgatcg ccatgacctg ttatttatgg
     3901 cacagacaaa accaagaacg ccgcaggatt gaaattagca tcaagcaaca aacccaacgg
     3961 gaacgattta ttaaccaaat tacccaacat atccgccaat ctttaaactt ggaaacggtt
     4021 ttaaatacca ccgtcgctga agttaaaacc ctgttgcaag ttgatcgagt tctaatttat
     4081 cgcatttggc aagatggcac gggcagcgcc attacggaat cggtgaatgc caattatcct
     4141 agtattttag ggcggacctt ttccgatgaa gtttttcccg ttgaatacca tcaagcctac
     4201 accaaaggta aagtacgggc cattaatgac attgaccagg atgacataga gatttgccta
     4261 gctgatttcg tcaaacaatt tggcgtgaaa tcaaaattag tagtgcccat tcttcaacat
     4321 aatcgtgctt cttccctaga taatgaatca gaatttccct atctttgggg gctgttaatt
     4381 acccatcaat gtgcttttac ccggccatgg caaccgtggg aagtggagtt aatgaaacag
     4441 ctagccaatc aggtcgcgat cgccatccaa caatcggaat tatatgagca attacagcaa
     4501 ctcaataaag atttggaaaa ccgagtcgaa aaacgcaccc agcaacttgc cgccaccaat
     4561 caatccctaa gaatggaaat cagtgagcga caaaaaacgg aagccgctct ccgccacact
     4621 aaccatactc tgcaatccct gattgcggcc tcccccaggg gtatttttac ccttaattta
     4681 gcagaccaaa ttcagatttg gaatcctaca gcagaacgta tttttggttg gacagaaaca
     4741 gaaattattg cccatccaga attattaaca tccaacattt tgctggaaga ttatcagcaa
     4801 tttaaacaga aagttttatc aggcatggtt tcccctagcc tagaattaaa atgtcaaaaa
     4861 aaagatggta gttggattga aattgtcctt tccgctgctc ccctattgga tagtgaagaa
     4921 aatattgccg gattggtggc ggttgtcgcc gatattaccg agcaaaagcg gcaggcataa
     4981 caaattcgtt tgctacaatc cgttgtggtt aatactaatg atgcggtggt gattacggaa
     5041 gcggagccca ttgatgatcc cgggccgaga attctctatg tcaatgaagc atttactaaa
     5101 atcaccggtt atactgctga agaaatgcta ggcaaaaccc cccgagtttt acagggacca
     5161 aaaactagtc gcactgaatt agatagggtg cggcaagcca ttagtcaatg gcaatcagtt
     5221 accgttgaag tgattaatta tcgtaaggat ggcagtgagt tttgggtgga atttagtctg
     5281 gtgcccgttg ccaataaaac aggtttttac acccattgga ttgctgtgca aagggatgtc
     5341 actgagcgcc gacgcacgga ggaagtccgc ctagctttag aacgggaaaa agaattaagc
     5401 cgcctaaaaa ctcgtttttt ctccatggct tcccatgaat ttcgtactcc cctcagtacg
     5461 gccttagctg ctgcccaatt actggaaaat tctgaagtgg cctggcttga tcccgataag
     5521 cgtagccgga acttacaccg tattcaaaat tccgtgaaaa atatggtaca gctcctggat
     5581 gatattttaa tcattaaccg tgccgaagcg ggcaaattgg aatttaatcc taattggtta
     5641 gatttgaaat tattgttcca gcaatttatc gaagaaattc aattaagtgt cagtgaccaa
     5701 tattattttg actttatttg tagcgctcaa gatacgaagg cattggtgga tgaaaggtta
     5761 gtgcggtcta ttttatctaa tctgttatct aatgcgatta aatactctcc cgggggaggg
     5821 cagattaaaa ttgccctaag cctagattcg gaacagatta tttttgaagt caccgaccag
     5881 ggcattggca tttcgccaga ggaccaaaag caaatttttg aaccctttca tcggggcaaa
     5941 aatgtcagaa atattacggg aacaggactc ggtttaatgg ttgccaagaa atgtgttgac
     6001 ttacacagtg gcagtatctt gctaaaaagt gcagttgacc agggaacaac agttactatc
     6061 tgtttaaaac gctataacca tttgcctcga gcttaggacg tcgatatctg gcgaaaatga
     6121 gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg
     6181 ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
     6241 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     6301 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     6361 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     6421 cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata
     6481 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
     6541 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
     6601 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
     6661 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
     6721 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
     6781 atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg
     6841 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tgatatcgag
     6901 ctcgcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt
     6961 cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagca ctagtcatga
     7021 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     7081 aaggatcttc
//
