LOCUS       sequence-171058-        4124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A weak promoter drives the expression of a hypersensitive luxR 
            mutant, which regulates expression of LVA-tagged GFP under the 
            control of a strong RBS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4124)
  AUTHORS   Magaraci MS, Bermudez JG, Yogish D, Pak DH, Mollov V, Tycko J, 
            Issadore D, Mannickarottu SG, Chow BY
  TITLE     Toolbox for Exploring Modular Gene Regulation in Synthetic Biology 
            Training.
  JOURNAL   ACS Synth Biol. 2016 Apr 25.
  PUBMED    27111289
REFERENCE   2  (bases 1 to 4124)
  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. 2016 Apr 25."
COMMENT     Sequence Label: BC-A1-013
FEATURES             Location/Qualifiers
     source          1..4124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..714
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1..714
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(31..59)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     654..674
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             715..747
                     /codon_start=1
                     /product="C-terminal peptide that mediates degradation in 
                     bacteria through the ClpXP and ClpAP proteases (McGinness 
                     et al., 2006)"
                     /label=ssrA tag (LVA)
                     /note="mutant LVA variant that confers accelerated 
                     degradation under some conditions (Andersen et al., 1998)"
                     /translation="AANDENYALVA"
     terminator      801..872
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      888..915
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      937..994
                     /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."
     primer_bind     complement(1018..1035)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1189..1777)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1269..1288)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1960..2054)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2075..2734)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2649..2668
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2896..2914
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2918..2961)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        2971..3005
                     /label=BBa_J23109
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.04"
     RBS             3012..3023
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      3835..3906
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3922..3949
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4107..4118
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
ORIGIN
        1 atgcgtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
       61 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga
      121 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
      181 gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca catgaaacag
      241 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc
      301 aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt
      361 aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa
      421 ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga
      481 atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
      541 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
      601 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt
      661 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaagctgca
      721 aacgacgaaa actacgcttt agtagcttaa taatctagac actgatagtg ctagtgtaga
      781 tcactactag agccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
      841 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
      901 gtgggccttt ctgcgtttat acccgggtac tagaggtccg gcaaaaaagg gcaaggtgtc
      961 accaccctgc cctttttctt taaaaccgaa aagattactt cgcgttatgc aggcttcctc
     1021 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1081 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1141 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccacaggct
     1201 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1261 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1321 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1381 tcatagctca cgctgtaagt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1441 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1501 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1561 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1621 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1681 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1741 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1801 ggggtcagac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1861 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1921 tatatatgag taaacttggt ctgacagctc gaggcttgga ttctcaccaa taaaaaacgc
     1981 ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc
     2041 tatcaacagg agtccaagcg agctcgatat caaattacgc cccgccctgc cactcatcgc
     2101 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
     2161 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
     2221 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     2281 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     2341 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     2401 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     2461 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt
     2521 ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
     2581 tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
     2641 tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
     2701 caacggtggt atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
     2761 tcgataactc aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac
     2821 ctcttacgtg cccgatcaac tcgagtgcca cctgacgtct aagaaaccat tattatcatg
     2881 acattaacct ataaaaatag gcgtatcacg aggcagaatt tcagataaaa aaaatcctta
     2941 gctttcgcta aggatgattt ctggctgcag tttacagcta gctcagtcct agggactgtg
     3001 ctagcggatc caaagaggag aaagaattca tgaaaaacat aaatgccgac gacacataca
     3061 gaataattaa taaaattaaa gcgtgtagaa gcaataatga tattaatcaa tgcttatctg
     3121 atatggctaa aatggtacat tgtgaatatt atttactcgc gttcatttat cctcattcta
     3181 tggttaaatc agatatttca atcctagata attaccctaa aaaatggagg caatattatg
     3241 atgacgctaa tttaataaaa tatgatccta tagtagatta ttctaactcc aatcattcac
     3301 caattaattg gaatatattt gaaaacaatg ctgtaaataa aaaatctcca aatgtaatta
     3361 aagaagcgaa aacagcaggt cttatcactg ggtttagttt ccctattcat acggctaaca
     3421 atggcttcgg aatgcttagt tttgcacatt cagaaaaaga caactatata gatagtttat
     3481 ttttacatgc gtgtatgaac ataccattaa ttgttccttc tctagttgat aattatcgaa
     3541 aaataaatat agcaaataat aaatcaaaca acgatttaac caaaagagaa aaagaatgtt
     3601 tagcgtgggc atgcgaagga aaaagctctt gggatatttc aaaaatatta ggttgcagtg
     3661 agcgtactgt cactttccat ttaaccaatg cgcaaatgaa actcaataca acaaaccgct
     3721 gccaaagtat ttctaaagca attttaacag gagcaattga ttgcccatac tttaaaaatt
     3781 aataagcggc cgccactgat agtgctagtg tagatcacta ctagagccag gcatcaaata
     3841 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     3901 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttataggtac
     3961 ctactagaga ggttctgtta agtaactgaa cccaatgtcg ttagtgacgc ttacctctta
     4021 agaggtcact gacctaacaa agcttacctg taggatcgta caggtttacg caagaaaatg
     4081 gtttgttata gtcgaataaa actagtaaag aggagaaata gcat
//
