LOCUS       sequence-274938-        4601 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Natural Terminator ECK120033736 inserted between mCherry and GFP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4601)
  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 4601)
  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 464
FEATURES             Location/Qualifiers
     source          1..4601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             24..683
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(90..109)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     oriT            832..940
                     /note="incP origin of transfer"
     CDS             complement(951..1613)
                     /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      1614..2385
                     /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(2500..2586)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2601..2623
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        2631..2665
                     /label=BBa_J23104
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.72"
     RBS             2673..2684
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2691..3401
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2836..2854)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3104..3123
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     RBS             3480..3491
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3498..4211
                     /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(3528..3556)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4151..4171
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4234..4305
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4321..4348
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    4355..4375
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      4380..4474
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(4522..4601,1..23)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
       61 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
      121 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
      181 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
      241 ttcgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
      301 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
      361 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
      421 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
      481 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
      541 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
      601 atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
      661 atgagtggca gggcggggcg taatttgact tttgtccttt tccgctgcat aaccctgctt
      721 cggggtcatt atagcgattt tttcggtata tccatccttt ttcgcacgat atacaggatt
      781 ttgccaaagg gttcgtgtag actttccttg gtgtatccaa cggcgtcagc cgggcaggat
      841 aggtgaagta ggcccacccg cgagcgggtg ttccttcttc actgtccctt attcgcacct
      901 ggcggtgctc aacgggaatc ctgctctgcg aggctggccg taggccggcc ctaccggcgc
      961 ggcagcgtta cccgtgtcgg cggctccaac ggctcgccat cgtccagaaa acacggctca
     1021 tcgggcatcg gcaggcgctg ctgcccgcgc cgttcccatt cctccgtttc ggtcaaggct
     1081 ggcaggtctg gttccatgcc cggaatgccg ggctggctgg gcggctcctc gccggggccg
     1141 gtcggtagtt gctgctcgcc cggatacagg gtcgggatgc ggcgcaggtc gccatgcccc
     1201 aacagcgatt cgtcctggtc gtcgtgatca accaccacgg cggcactgaa caccgacagg
     1261 cgcaactggt cgcggggctg gccccacgcc acgcggtcat tgaccacgta ggccgacacg
     1321 gtgccggggc cgttgagctt cacgacggag atccagcgct cggccaccaa gtccttgact
     1381 gcgtattgga ccgtccgcaa agaacgtccg atgagcttgg aaagtgtctt ctggctgacc
     1441 accacggcgt tctggtggcc catctgcgcc acgaggtgat gcagcagcat tgccgccgtg
     1501 ggtttcctcg caataagccc ggcccacgcc tcatgcgctt tgcgttccgt ttgcacccag
     1561 tgaccgggct tgttcttggc ttgaatgccg atttctctgg actgcgtggc catgcttatc
     1621 tccatgcggt aggggtgccg cacggttgcg gcaccatgcg caatcagctg caacttttcg
     1681 gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga ttttctttaa
     1741 cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc cccctttttt
     1801 aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt ggtgcccaaa
     1861 gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc cggcaaaaag
     1921 tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca aggtggcgct
     1981 gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg ggcgggcttc
     2041 gcccttcgac tgcccccact cgcataggct tgggtcgttc caggcgcgtc aaggccaagc
     2101 cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac ttggtgtcca accggcaagc
     2161 gaagcgcgca ggccgcaggc cggaggcttt tccccagaga aaattaaaaa aattgatggg
     2221 gcaaggccgc aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc tgggtagccg
     2281 gtgggcaatc cctgtggtca agctcgtggg caggcgcagc ctgtccatca gcttgtccag
     2341 cagggttgtc cacgggccga gcgaagcgag ccagccggtg gccgctcgcg gccatcgtcc
     2401 acatatccac gggctggcaa gggagcgcag cgaccgcgca gggcgaagcc cggagagcaa
     2461 gcccgtaggg ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac tcaggagagc
     2521 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     2581 tatttgatgc ctttaattaa agcggataac aatttcacac aggagaattc ttgacagcta
     2641 gctcagtcct aggtattgtg ctagctctag agaaagagga gaaatactag atggtgagca
     2701 agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     2761 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2821 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     2881 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     2941 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     3001 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cttgcaggac ggcgagttca
     3061 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     3121 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     3181 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     3241 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     3301 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     3361 gccgccactc caccggcggc atggacgagc tgtacaagta ataatacgga tccaacgcat
     3421 gagaaagccc ccggaagatc accttccggg ggctttttta ttgcgccctg caggtagaga
     3481 aagaggagaa atactagatg cgtaaaggag aagaactttt cactggagtt gtcccaattc
     3541 ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag
     3601 gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg
     3661 ttccatggcc aacacttgtc actactttcg gttatggtgt tcaatgcttt gcgagatacc
     3721 cagatcatat gaaacagcat gactttttca agagtgccat gcccgaaggt tatgtacagg
     3781 aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg
     3841 aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa
     3901 acattcttgg acacaaattg gaatacaact ataactcaca caatgtatac atcatggcag
     3961 acaaacaaaa gaatggaatc aaagttaact tcaaaattag acacaacatt gaagatggaa
     4021 gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt
     4081 taccagacaa ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga
     4141 gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg
     4201 aactatacaa ataataatac tagagccagg catcaaataa aacgaaaggc tcagtcgaaa
     4261 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     4321 ggctcacctt cgggtgggcc tttctgcgtt tatatactag tagcggccgc tgcagcttgg
     4381 actcctgttg atagatccag taatgacctc agaactccat ctggatttgt tcagaacgct
     4441 cggttgccgc cgggcgtttt ttattggtga gaatccaggg gtccccaata attacgattt
     4501 aaattggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga
     4561 aataagatca ctaccgggcg tattttttga gttatcgaga t
//
