LOCUS       sequence-495670-        7011 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  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 7011)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 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..7011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1023..1794
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             1795..2457
                     /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     3151..3173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3165..3182
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3166..3182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3192..3211
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3192..3210
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3253..3339
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3436..3522
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3619..3705
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3802..3888
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3985..4071
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    4163..4219
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             4270..4983
                     /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(4300..4328)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4923..4943
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5004..5090
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5202..5222)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5202..5220)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5241..5257)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5241..5257)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5254..5276)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5265..5281
                     /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(5289..5295,5296..5313,5314..5319))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5334..5355
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(5760..6554)
                     /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(5871..5890)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6481..6500
                     /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 gaagggcgaa gcccgcccgc
     1381 ctgccccccg agcctcacgg cggcgagtgc gggggttcca agggggcagc gccaccttgg
     1441 gcaaggccga aggccgcgca gtcgatcaac aagccccgga ggggccactt tttgccggag
     1501 ggggagccgc gccgaaggcg tgggggaacc ccgcaggggt gcccttcttt gggcaccaaa
     1561 gaactagata tagggcgaaa tgcgaaagac ttaaaaatca acaacttaaa aaaggggggt
     1621 acgcaacagc tcattgcggc accccccgca atagctcatt gcgtaggtta aagaaaatct
     1681 gtaattgact gccactttta cgcaacgcat aattgttgtc gcgctgccga aaagttgcag
     1741 ctgattgcgc atggtgccgc aaccgtgcgg cacccctacc gcatggagat aagcatggcc
     1801 acgcagtcca gagaaatcgg cattcaagcc aagaacaagc ccggtcactg ggtgcaaacg
     1861 gaacgcaaag cgcatgaggc gtgggccggg cttattgcga ggaaacccac ggcggcaatg
     1921 ctgctgcatc acctcgtggc gcagatgggc caccagaacg ccgtggtggt cagccagaag
     1981 acactttcca agctcatcgg acgttctttg cggacggtcc aatacgcagt caaggacttg
     2041 gtggccgagc gctggatctc cgtcgtgaag ctcaacggcc ccggcaccgt gtcggcctac
     2101 gtggtcaatg accgcgtggc gtggggccag ccccgcgacc agttgcgcct gtcggtgttc
     2161 agtgccgccg tggtggttga tcacgacgac caggacgaat cgctgttggg gcatggcgac
     2221 ctgcgccgca tcccgaccct gtatccgggc gagcagcaac taccgaccgg ccccggcgag
     2281 gagccgccca gccagcccgg cattccgggc atggaaccag acctgccagc cttgaccgaa
     2341 acggaggaat gggaacggcg cgggcagcag cgcctgccga tgcccgatga gccgtgtttt
     2401 ctggacgatg gcgagccgtt ggagccgccg acacgggtca cgctgccgcg ccggtagcac
     2461 ttgggttgcg cagcaacccg taagtgcgct gttccagact atcggctgta gccgcctcgc
     2521 cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc
     2581 gacctgaatg gaagccggcg gcacctcgct aacggattca ccgtttttat caggctctgg
     2641 gaggcagaat aaatgatcat atcgtcaatt attacctcca cggggagagc ctgagcaaac
     2701 tggcctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     2761 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg
     2821 aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac
     2881 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
     2941 cgcagtacgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3001 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
     3061 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3181 gtgagcgcgc gtaatacgac tcactatagg gcgaattgga gctggggccg caattcccaa
     3241 ttccccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     3301 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     3361 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggaat
     3421 taattcccca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3481 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     3541 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     3601 aattaattcc ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     3661 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     3721 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     3781 aggaattaat tccccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     3841 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     3901 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     3961 gccaggaatt aattccccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4021 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     4081 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     4141 actgccagga attggggatc ggaagcttgc atgcctgcag gtcgactcta gaggatcccc
     4201 gggtaccgag ctcgaattcg gcttattccc taactaacta aagattaact ttataaggag
     4261 gaaaaacata tgagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa
     4321 ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca
     4381 acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg
     4441 ccaacacttg tcactacttt gacttatggt gttcaatgct tttcaagata cccagatcat
     4501 atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact
     4561 atatttttca aagatgacgg gaactataag acacgtgctg aagtcaagtt tgaaggtgat
     4621 acacttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt
     4681 ggacacaagt tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa
     4741 aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa
     4801 ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac
     4861 aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac
     4921 atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac
     4981 aaataggaat taattccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     5041 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     5101 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     5161 ctgccaggaa ttgggggcgg ccccgtaccc agcttttgtt ccctttagtg agggttaatt
     5221 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5281 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5341 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5401 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5461 atgcataaaa actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
     5521 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
     5581 cccatggggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc
     5641 ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa
     5701 atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct
     5761 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
     5821 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
     5881 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
     5941 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
     6001 acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg
     6061 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
     6121 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
     6181 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
     6241 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     6301 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     6361 gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     6421 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     6481 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     6541 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatct tgatcccctg
     6601 cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc
     6661 ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag
     6721 tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt
     6781 tcccttgtcc agatagccca gtagctgaca ttcatcccag gtggcacttt tcggggaaat
     6841 gtgcgcgccc gcgttcctgc tggcgctggg cctgtttctg gcgctggact tcccgctgtt
     6901 ccgtcagcag cttttcgccc acggccttga tgatcgcggc ggccttggcc tgcatatccc
     6961 gattcaacgg ccccagggcg tccagaacgg gcttcaggcg ctcccgaagg t
//
