LOCUS       sequence-131392-        6816 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 6816)
  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 6816)
  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..6816
                     /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"
     misc_feature    3968..4024
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     CDS             4075..4788
                     /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(4105..4133)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4728..4748
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4809..4895
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5007..5027)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5007..5025)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5046..5062)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5046..5062)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5059..5081)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5070..5086
                     /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(5094..5100,5101..5118,5119..5124))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5139..5160
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(5565..6359)
                     /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(5676..5695)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6286..6305
                     /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 cttgcatgcc tgcaggtcga ctctagagga tccccgggta ccgagctcga
     4021 attcggctta ttccctaact aactaaagat taactttata aggaggaaaa acatatgagt
     4081 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt
     4141 aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt
     4201 acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact
     4261 actttgactt atggtgttca atgcttttca agatacccag atcatatgaa acggcatgac
     4321 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat
     4381 gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga
     4441 atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa
     4501 tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa tggaatcaaa
     4561 gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc agaccattat
     4621 caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcc
     4681 acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag
     4741 tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata ggaattaatt
     4801 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     4861 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     4921 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattggg
     4981 ggcggccccg tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     5041 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5101 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5161 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5221 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgcatgca taaaaactgt
     5281 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     5341 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat gggggtgggc
     5401 gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac
     5461 gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag
     5521 gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca
     5581 agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg
     5641 aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg
     5701 tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca
     5761 ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg
     5821 tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct
     5881 tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg
     5941 cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc
     6001 attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc
     6061 tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc
     6121 acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc
     6181 agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct
     6241 gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg
     6301 aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg
     6361 cgaaacgatc ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt
     6421 ggcggcaaga aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc
     6481 ccagctggca attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat
     6541 gtaagcccac tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata
     6601 gcccagtagc tgacattcat cccaggtggc acttttcggg gaaatgtgcg cgcccgcgtt
     6661 cctgctggcg ctgggcctgt ttctggcgct ggacttcccg ctgttccgtc agcagctttt
     6721 cgcccacggc cttgatgatc gcggcggcct tggcctgcat atcccgattc aacggcccca
     6781 gggcgtccag aacgggcttc aggcgctccc gaaggt
//
