LOCUS       sequence-274841-        3831 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes Ribosome Binding Site (RBS) variant using GFP as a reporter 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3831)
  AUTHORS   Teh MY, Ooi KH, Danny Teo SX, Bin Mansoor ME, Shaun Lim WZ, Tan MH
  TITLE     An Expanded Synthetic Biology Toolkit for Gene Expression Control in
            Acetobacteraceae.
  JOURNAL   ACS Synth Biol. 2019 Apr 19;8(4):708-723. doi: 
            10.1021/acssynbio.8b00168. Epub 2019 Mar 22.
  PUBMED    30865830
REFERENCE   2  (bases 1 to 3831)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acssynbio.8b00168"; journalName: "ACS Synth Biol"; date: 
            "2019-04-19- 19"; volume: "8"; issue: "4"; pages: "708-723"
COMMENT     Sequence Label: MYMH 25
FEATURES             Location/Qualifiers
     source          1..3831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      4..98
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        146..248
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             249..908
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(315..334)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     oriT            1057..1165
                     /note="incP origin of transfer"
     CDS             complement(1176..1838)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      1839..2610
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(2725..2811)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2826..2848
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    2850..2871
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     protein_bind    2874..2890
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    2897..2913
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             2953..3666
                     /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"
     primer_bind     complement(2983..3011)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3606..3626
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3689..3760
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3776..3803
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    3810..3830
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
ORIGIN
        1 ttggactcct gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa
       61 cgctcggttg ccgccgggcg ttttttattg gtgagaatcc aggggtcccc aataattacg
      121 atttaaattg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata
      181 atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg
      241 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
      301 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
      361 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
      421 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt atggcaatga
      481 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc
      541 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
      601 acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt
      661 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
      721 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
      781 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
      841 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg
      901 gggcgtaatt tgacttttgt ccttttccgc tgcataaccc tgcttcgggg tcattatagc
      961 gattttttcg gtatatccat cctttttcgc acgatataca ggattttgcc aaagggttcg
     1021 tgtagacttt ccttggtgta tccaacggcg tcagccgggc aggataggtg aagtaggccc
     1081 acccgcgagc gggtgttcct tcttcactgt cccttattcg cacctggcgg tgctcaacgg
     1141 gaatcctgct ctgcgaggct ggccgtaggc cggccctacc ggcgcggcag cgttacccgt
     1201 gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg catcggcagg
     1261 cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag gtctggttcc
     1321 atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc
     1381 tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag cgattcgtcc
     1441 tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg
     1501 ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc ggggccgttg
     1561 agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta ttggaccgtc
     1621 cgcaaagaac gtccgatgag cttggaaagt gtcttctggc tgaccaccac ggcgttctgg
     1681 tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt cctcgcaata
     1741 agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc gggcttgttc
     1801 ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat gcggtagggg
     1861 tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc gcgacaacaa
     1921 ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac gcaatgagct
     1981 attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt gttgattttt
     2041 aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg gcacccctgc
     2101 ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc cctccggggc
     2161 ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc cttggaaccc
     2221 ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgccct tcgactgccc
     2281 ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct
     2341 gcgcgagcct tgacccgcct tccacttggt gtccaaccgg caagcgaagc gcgcaggccg
     2401 caggccggag gcttttcccc agagaaaatt aaaaaaattg atggggcaag gccgcaggcc
     2461 gcgcagttgg agccggtggg tatgtggtcg aaggctgggt agccggtggg caatccctgt
     2521 ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg tccagcaggg ttgtccacgg
     2581 gccgagcgaa gcgagccagc cggtggccgc tcgcggccat cgtccacata tccacgggct
     2641 ggcaagggag cgcagcgacc gcgcagggcg aagcccggag agcaagcccg tagggggggc
     2701 gcgcccagct gtctagggcg gcggatttgt cctactcagg agagcgttca ccgacaaaca
     2761 acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgccttta
     2821 attaaagcgg ataacaattt cacacaggag aattcgcggc cgcttctaga gaattgtgag
     2881 cggataacaa ttgacattgt gagcggataa caagatactg agcacatact agagaaaaag
     2941 gagaaatact agatgcgtaa aggagaagaa cttttcactg gagttgtccc aattcttgtt
     3001 gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
     3061 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca
     3121 tggccaacac ttgtcactac tttcggttat ggtgttcaat gctttgcgag atacccagat
     3181 catatgaaac agcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga
     3241 actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt
     3301 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
     3361 cttggacaca aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa
     3421 caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt
     3481 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
     3541 gacaaccatt acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac
     3601 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta
     3661 tacaaataat aatactagag ccaggcatca aataaaacga aaggctcagt cgaaagactg
     3721 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     3781 accttcgggt gggcctttct gcgtttatat actagtagcg gccgctgcag c
//
