LOCUS       sequence-47441-e        6810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6810)
  AUTHORS   Miller WG, Leveau JH, Lindow SE
  TITLE     Improved gfp and inaZ broad-host-range promoter-probe vectors.
  JOURNAL   Mol Plant Microbe Interact. 2000 Nov;13(11):1243-50.
  PUBMED    11059491
REFERENCE   2  (bases 1 to 6810)
  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: "Mol Plant 
            Microbe Interact."; date: "2000-11"; volume: "13(11)"; pages: 
            "1243-50"
COMMENT     Sequence Label: pPROBE-NT'
FEATURES             Location/Qualifiers
     source          1..6810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1023..1792
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             1793..2455
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     primer_bind     3147..3169
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3161..3178
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3162..3178
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3188..3207
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3188..3206
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3247..3333
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3428..3514
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3609..3695
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3790..3876
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             4069..4782
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4099..4127)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4722..4742
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4803..4889
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5001..5021)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5001..5019)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5040..5056)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5040..5056)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5053..5075)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5064..5080
                     /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)."
     promoter        complement(join(5088..5094,5095..5112,5113..5118))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5133..5154
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(5559..6353)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5670..5689)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6280..6299
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 ctcgggccgt ctcttgggct tgatcggcct tcttgcgcat ctcacgcgct cctgcggcgg
       61 cctgtagggc aggctcatac ccctgccgaa ccgcttttgt cagccggtcg gccacggctt
      121 ccggcgtctc aacgcgcttt gagattccca gcttttcggc caatccctgc ggtgcatagg
      181 cgcgtggctc gaccgcttgc gggctgatgg tgacgtggcc cactggtggc cgctccaggg
      241 cctcgtagaa cgcctgaatg cgcgtgtgac gtgccttgct gccctcgatg ccccgttgca
      301 gccctagatc ggccacagcg gccgcaaacg tggtctggtc gcgggtcatc tgcgctttgt
      361 tgccgatgaa ctccttggcc gacagcctgc cgtcctgcgt cagcggcacc acgaacgcgg
      421 tcatgtgcgg gctggtttcg tcacggtgga tgctggccgt cacgatgcga tccgccccgt
      481 acttgtccgc cagccacttg tgcgccttct cgaagaacgc cgcctgctgt tcttggctgg
      541 ccgacttcca ccattccggg ctggccgtca tgacgtactc gaccgccaac acagcgtcct
      601 tgcgccgctt ctctggcagc aactcgcgca gtcggcccat cgcttcatcg gtgctgctgg
      661 ccgcccagtg ctcgttctct ggcgtcctgc tggcgtcagc gttgggcgtc tcgcgctcgc
      721 ggtaggcgtg cttgagactg gccgccacgt tgcccatttt cgccagcttc ttgcatcgca
      781 tgatcgcgta tgccgccatg cctgcccctc ccttttggtg tccaaccggc tcgacggggg
      841 cagcgcaagg cggtgcctcc ggcgggccac tcaatgcttg agtatactca ctagactttg
      901 cttcgcaaag tcgtgaccgc ctacggcggc tgcggcgccc tacgggcttg ctctccgggc
      961 ttcgccctgc gcggtcgctg cgctcccttg ccagcccgtg gatatgtgga cgatggccgc
     1021 gagcggccac cggctggctc gcttcgctcg gcccgtggac aaccctgctg gacaagctga
     1081 tggacaggct gcgcctgccc acgagcttga ccacagggat tgcccaccgg ctacccagcc
     1141 ttcgaccaca tacccaccgg ctccaactgc gcggcctgcg gccttgcccc atcaattttt
     1201 ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct tgccggttgg
     1261 acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct tggccttgac
     1321 gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaaggcgaag cccgcccgcc
     1381 tgccccccga gcctcacggc ggcgagtgcg ggggttccaa gggggcagcg ccaccttggg
     1441 caaggccgaa ggccgcgcag tcgatcaaca agccccggag gggccacttt ttgccggagg
     1501 gggagccgcg ccgaaggcgt gggggaaccc cgcaggggtg cccttctttg ggcaccaaag
     1561 aactagatat agggcgaaat gcgaaagact taaaaatcaa caacttaaaa aaggggggta
     1621 cgcaacagct cattgcggca ccccccgcaa tagctcattg cgtaggttaa agaaaatctg
     1681 taattgactg ccacttttac gcaacgcata attgttgtcg cgctgccgaa aagttgcagc
     1741 tgattgcgca tggtgccgca accgtgcggc accctaccgc atggagataa gcatggccac
     1801 gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga
     1861 acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct
     1921 gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaagac
     1981 actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt
     2041 ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt
     2101 ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag
     2161 tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct
     2221 gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga
     2281 gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac
     2341 ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct
     2401 ggacgatggc gagccgttgg agccgccgac acgggtcacg ctgccgcgcc ggtagcactt
     2461 gggttgcgca gcaacccgta agtgcgctgt tccagactat cggctgtagc cgcctcgccg
     2521 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     2581 cctgaatgga agccggcggc acctcgctaa cggattcacc gtttttatca ggctctggga
     2641 ggcagaataa atgatcatat cgtcaattat tacctccacg gggagagcct gagcaaactg
     2701 gcctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     2761 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa
     2821 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaccag
     2881 gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc
     2941 agtcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3001 aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg
     3061 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     3121 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     3181 gcgcgcgtaa tacgactcac tatagggcga attggagctg gggccgcaat tcccaattcc
     3241 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     3301 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     3361 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gaattaattc
     3421 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     3481 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
     3541 gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca ggaattaatt
     3601 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     3661 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     3721 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattaat
     3781 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     3841 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt
     3901 gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggaattgg
     3961 ggatcggaag ctgaattcga gctcaggcct tggtacccgg gatccgtcga ctgcagaagc
     4021 ttaattccct aactaactaa agattaactt tataaggagg aaaaacatat gagtaaagga
     4081 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
     4141 cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt
     4201 aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttg
     4261 acttatggtg ttcaatgctt ttcaagatac ccagatcata tgaaacggca tgactttttc
     4321 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg
     4381 aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag
     4441 ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac
     4501 tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac
     4561 ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
     4621 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa
     4681 tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta
     4741 acagctgctg ggattacaca tggcatggat gaactataca aataggaatt aattccaggc
     4801 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     4861 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc
     4921 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat tgggggcggc
     4981 cccgtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     5041 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5101 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5161 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5221 ggccaacgcg cggggagagg cggtttgcgt attgggcgca tgcataaaaa ctgttgtaat
     5281 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     5341 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatgggggt gggcgaagaa
     5401 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
     5461 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
     5521 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
     5581 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     5641 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     5701 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     5761 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     5821 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     5881 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     5941 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     6001 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     6061 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     6121 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca
     6181 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     6241 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     6301 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     6361 gatcctcatc ctgtctcttg atcagatctt gatcccctgc gccatcagat ccttggcggc
     6421 aagaaagcca tccagtttac tttgcagggc ttcccaacct taccagaggg cgccccagct
     6481 ggcaattccg gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc
     6541 ccactgcaag ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag
     6601 tagctgacat tcatcccagg tggcactttt cggggaaatg tgcgcgcccg cgttcctgct
     6661 ggcgctgggc ctgtttctgg cgctggactt cccgctgttc cgtcagcagc ttttcgccca
     6721 cggccttgat gatcgcggcg gccttggcct gcatatcccg attcaacggc cccagggcgt
     6781 ccagaacggg cttcaggcgc tcccgaaggt
//
