LOCUS       sequence-388746-        3770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP under the control of J23100 and RiboJ-BCD2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 5 to 39)
  AUTHORS   Keating KW, Young EM
  TITLE     Systematic Part Transfer by Extending a Modular Toolkit to Diverse 
            Bacteria.
  JOURNAL   ACS Synth Biol. 2023 Jun 9. doi: 10.1021/acssynbio.3c00104.
  PUBMED    37294017
REFERENCE   2  (bases 1 to 3770)
  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. 2023 Jun 9. doi: 10.1021/acssynbio.3c00104."
COMMENT     Sequence Label: pBBR1_DVK_AE(J23100-RiboJ-BCD2-E0040m-B0015)
FEATURES             Location/Qualifiers
     source          1..3770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        5..39
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     misc_RNA        46..95
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             189..197
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             207..920
                     /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(237..265)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     860..880
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      939..1010
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1026..1053
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      1201..1972
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             1973..2635
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     oriT            2646..2754
                     /note="incP origin of transfer"
     primer_bind     complement(2844..2863)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2855..3670
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2855..3670
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2928..2947)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ggagttgacg gctagctcag tcctaggtac agtgctagct actagctgtc accggatgtg
       61 ctttccggtc tgatgagtcc gtgaggacga aacagcctct acaaataatt ttgtttaagg
      121 gcccaagttc acttaaaaag gagatcaaca atgaaagcaa ttttcgtact gaaacatctt
      181 aatcatgcta aggaggtttt ctaatgatgc gtaaaggaga agaacttttc actggagttg
      241 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
      301 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
      361 aactacctgt tccatggcca acacttgtca ctactttcgg ttatggtgtt caatgctttg
      421 cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt
      481 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
      541 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
      601 aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca
      661 tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg
      721 aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc
      781 ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca
      841 acgaaaagag agatcacatg gtccttcttg agtttgtaac agctgctggg attacacatg
      901 gcatggatga actatacaaa taataaaggt ccaggcatca aataaaacga aaggctcagt
      961 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     1021 acactggctc accttcgggt gggcctttct gcgtttatag cttatgtctt cagcagtagg
     1081 acaaatccgc cgccctagac agctgggcgc gcccccccta cgggcttgct ctccgggctt
     1141 cgccctgcgc ggtcgctgcg ctcccttgcc agcccgtgga tatgtggacg atggccgcga
     1201 gcggccaccg gctggctcgc ttcgctcggc ccgtggacaa ccctgctgga caagctgatg
     1261 gacaggctgc gcctgcccac gagcttgacc acagggattg cccaccggct acccagcctt
     1321 cgaccacata cccaccggct ccaactgcgc ggcctgcggc cttgccccat caattttttt
     1381 aattttctct ggggaaaagc ctccggcctg cggcctgcgc gcttcgcttg ccggttggac
     1441 accaagtgga aggcgggtca aggctcgcgc agcgaccgcg cagcggcttg gccttgacgc
     1501 gcctggaacg acccaagcct atgcgagtgg gggcagtcga agggcgaagc ccgcccgcct
     1561 gccccccgag cctcacggcg gcgagtgcgg gggttccaag ggggcagcgc caccttgggc
     1621 aaggccgaag gccgcgcagt cgatcaacaa gccccggagg ggccactttt tgccggaggg
     1681 ggagccgcgc cgaaggcgtg ggggaacccc gcaggggtgc ccttctttgg gcaccaaaga
     1741 actagatata gggcgaaatg cgaaagactt aaaaatcaac aacttaaaaa aggggggtac
     1801 gcaacagctc attgcggcac cccccgcaat agctcattgc gtaggttaaa gaaaatctgt
     1861 aattgactgc cacttttacg caacgcataa ttgttgtcgc gctgccgaaa agttgcagct
     1921 gattgcgcat ggtgccgcaa ccgtgcggca cccctaccgc atggagataa gcatggccac
     1981 gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga
     2041 acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct
     2101 gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaaaac
     2161 actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt
     2221 ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt
     2281 ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag
     2341 tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct
     2401 gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga
     2461 gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac
     2521 ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct
     2581 ggacgatggc gagccgttgg agccgccgac acgggtaacg ctgccgcgcc ggtagggccg
     2641 gcctacggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg
     2701 acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct gcccttgtgt
     2761 ctcaaaatct ctgatgtaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     2821 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     2881 ttgctccagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     2941 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     3001 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     3061 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     3121 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     3181 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     3241 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     3301 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     3361 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc
     3421 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     3481 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     3541 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     3601 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     3661 gtttttctaa ggtaactgtc agaccaagtt tactcatata tactttagat tgatttttcg
     3721 ttccactgag cgtcagaccc cagaactctg tcagaccgag tggaagacat
//
