LOCUS       sequence-313294-        6617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad-Host range expression vector, pBBR origin, TetR repressor, 
            PTetA promoter, GFPmut3b, Kanamycin Resistance Marker. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6617)
  AUTHORS   Schuster LA, Reisch CR
  TITLE     A plasmid toolbox for controlled gene expression across the 
            Proteobacteria.
  JOURNAL   Nucleic Acids Res. 2021 Jun 14. pii: 6298615. doi: 
            10.1093/nar/gkab496.
  PUBMED    34125913
REFERENCE   2  (bases 1 to 6617)
  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: "Nucleic 
            Acids Res. 2021 Jun 14. pii: 6298615. doi: 10.1093/nar/gkab496."
COMMENT     Sequence Label: pBTG2
FEATURES             Location/Qualifiers
     source          1..6617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1248..2019
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2020..2682
                     /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     3376..3398
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3390..3407
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3391..3407
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3512..4138)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4043..4062
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        join(4155..4160,4161..4178,4179..4183,4184..4189)
                     /label=tetA promoter
                     /note="contains a tet operator sequence that enables 
                     repression by TetR"
     protein_bind    4160..4178
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     misc_RNA        4202..4251
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             4296..4307
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4314..5027
                     /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(4344..4372)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4967..4987
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     misc_feature    5034..5054
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      5055..5112
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(5136..5153)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(5591..6385)
                     /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
                     VLFVKTDLSGALNELKDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5702..5721)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6312..6331
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gtccagatag cccagtagct gacattcatc ccaggtggca cttttcgtgg aaatgtgcgc
       61 gcccgcgttc ctgctggcgc tgggcctgtt tctggcgctg gacttcccgc tgttccgtca
      121 gcagcttttc gcccacggcc ttgatgatcg cggcggcctt ggcctgcata tcccgattca
      181 acggccccag ggcgtccaga acggtcttca ggcgctcccg aaggtctcgg gccgtctctt
      241 gggcttgatc ggccttcttg cgcatctcac gcgctcctgc ggcggcctgt agggcaggct
      301 catacccctg ccgaaccgct tttgtcagcc ggtcggccac ggcttccggc gtctcaacgc
      361 gctttgagat tcccagcttt tcggccaatc cctgcggtgc ataggcgcgt ggctcgaccg
      421 cttgcgggct gatggtgacg tggcccactg gtggccgctc cagggcctcg tagaacgcct
      481 gaatgcgcgt gtgacgtgcc ttgctgccct cgatgccccg ttgcagccct agatcggcca
      541 cagcggccgc aaacgtggtc tggtcgcggg tcatctgcgc tttgttgccg atgaactcct
      601 tggccgacag cctgccgtcc tgcgtcagcg gcaccacgaa cgcggtcatg tgcgggctgg
      661 tttcgtcacg gtggatgctg gccgtcacga tgcgatccgc cccgtacttg tccgccagcc
      721 acttgtgcgc cttctcgaag aacgccgcct gctgttcttg gctggccgac ttccaccatt
      781 ccgggctggc cgtcatgacg tactcgaccg ccaacacagc gtccttgcgc cgcttctctg
      841 gcagcaactc gcgcagtcgg cccatcgctt catcggtgct gctggccgcc cagtgctcgt
      901 tctctggcgt cctgctggcg tcagcgttgg gcgtctcgcg ctcgcggtag gcgtgcttga
      961 gactggccgc cacgttgccc attttcgcca gcttcttgca tcgcatgatc gcgtatgccg
     1021 ccatgcctgc ccctcccttt tggtgtccaa ccggctcgac gggggcagcg caaggcggtg
     1081 cctccggcgg gccactcaat gcttgagtat actcactaga ctttgcttcg caaagtagtg
     1141 accgcctacg gcggctgcgg cgccctacgg gcttgctctc agggcttcgc cctgcgcggt
     1201 cgctgcgctc ccttgccagc ccgtggatat gtggacgatg gccgcgagcg gccaccggct
     1261 ggctcgcttc gctcggcccg tggacaaccc tgctggacaa gctgatggac aggctgcgcc
     1321 tgcccacgag cttgaccaca gggattgccc accggctacc cagccttcga ccacataccc
     1381 accggctcca actgcgcggc ctgcggcctt gccccatcaa tttttttaat tttctctggg
     1441 gaaaagcctc cggcctgcgg cctgcgcgct tcgcttgccg gttggacacc aagtggaagg
     1501 cgggtcaagg ctcgcgcagc gaccgcgcag cggcttggcc ttgacgcgcc tggaacgacc
     1561 caagcctatg cgagtggggg cagtcgaagg gcgaagcccg cccgcctgcc ccccgagcct
     1621 cacggcggcg agtgcggggg ttccaagggg gcagcgccac cttgggcaag gccgaaggcc
     1681 gcgcagtcga tcaacaagcc ccggaggggc cactttttgc cggaggggga gccgcgccga
     1741 aggcgtgggg gaaccccgca ggggtgccct tctttgggca ccaaagaact agatataggg
     1801 cgaaatgcga aagacttaaa aatcaacaac ttaaaaaagg ggggtacgca acagctcatt
     1861 gcggcacccc ccgcaatagc tcattgcgta ggttaaagaa aatctgtaat tgactgccac
     1921 ttttacgcaa cgcataattg ttgtcgcgct gccgaaaagt tgcagctgat tgcgcatggt
     1981 gccgcaaccg tgcggcaccc ctaccgcatg gagataagca tggccacgca gtccagagaa
     2041 atcggcattc aagccaagaa caagcccggt cactgggtgc aaacggaacg caaagcgcat
     2101 gaggcgtggg ccgggcttat tgcgaggaaa cccacggcgg caatgctgct gcatcacctc
     2161 gtggcgcaga tgggccacca gaacgccgtg gtggtcagcc agaagacact ttccaagctc
     2221 atcggacgtt ctttgcggac ggtccaatac gcagtcaagg acttggtggc cgagcgctgg
     2281 atctccgtcg tgaagctcaa cggccccggc accgtgtcgg cctacgtggt caatgaccgc
     2341 gtggcgtggg gccagccccg cgaccagttg cgcctgtcgg tgttcagtgc cgccgtggtg
     2401 gttgatcacg acgaccagga cgaatcgctg ttggggcatg gcgacctgcg ccgcatcccg
     2461 accctgtatc cgggcgagca gcaactaccg accggccccg gcgaggagcc gcccagccag
     2521 cccggcattc cgggcatgga accagacctg ccagccttga ccgaaacgga ggaatgggaa
     2581 cggcgcgggc agcagcgcct gccgatgccc gatgagccgt gttttctgga cgatggcgag
     2641 ccgttggagc cgccgacacg ggtcacgctg ccgcgccggt agcacttggg ttgcgcagca
     2701 acccgtaagt gcgctgttcc agactatcgg ctgtagccgc ctcgccgccc tataccttgt
     2761 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc
     2821 cggcggcacc tcgctaacgg attcaccgtt tttatcaggc tctgggaggc agaataaatg
     2881 atcatatcgt caattattac ctccacgggg agagcctgag caaactggcc tcaggcattt
     2941 gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca
     3001 taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat
     3061 ttctgccatt catccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc
     3121 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tacggcctat
     3181 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3241 cttacaattt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     3301 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     3361 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgac ttctgttgag
     3421 ttaagaacga gtatcgagat ggcacatagc cttgctcaaa ttggaatcag gtttgtgcca
     3481 ataccagtag aaacagacga agaatccatg gttaagaccc actttcacat ttaagttgtt
     3541 tttctaatcc gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt
     3601 gatcaaataa ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt
     3661 ccctttcttc tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat
     3721 gccccacagc gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa
     3781 aggctaattg attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta
     3841 cttttgctcc atcgcgatga cttagtaaag cacatctaaa acttttagcg ttattacgta
     3901 aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca
     3961 tctcaatggc taaggcgtcg agcaaagccc gcttattttt tacatgccaa tacaatgtag
     4021 gctgctctac acctagcttc tgggcgagtt tacgggttgt taaaccttcg attccgacct
     4081 cattaagcag ctctaatgcg ctgttaatca ctttactttt atctaatcta gacatcatta
     4141 attcctaatt tttgttgaca ctctatcatt gatagagtta ttttaccact ccctatcaga
     4201 gctgtcaccg gatgtgcttt ccggtctgat gagtccgtga ggacgaaaca gcctctacaa
     4261 ataattttgt ttaagaactg ggaagagcgc agattaaaga ggagaaatac tagatgcgta
     4321 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta
     4381 atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta
     4441 cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta
     4501 ctttcggtta tggtgttcaa tgctttgcga gatacccaga tcatatgaaa cagcatgact
     4561 ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg
     4621 acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa
     4681 tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat
     4741 acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag
     4801 ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc
     4861 aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca
     4921 cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt
     4981 ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaataa taatactagt
     5041 agcggccgct gcagtccggc aaaaaagggc aaggtgtcac caccctgccc tttttcttta
     5101 aaaccgaaaa gattacttcg cgttatgcag gcttcctcgc tcactgactc gctgcgctcg
     5161 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatgct ttccagtcgg
     5221 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5281 gtattgggcg catgcataaa aactgttgta attcattaag cattctgccg acatggaagc
     5341 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg
     5401 tataatattt gcccatgggg gtgggcgaag aactccagca tgagatcccc gcgctggagg
     5461 atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg
     5521 gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc
     5581 agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
     5641 gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag
     5701 caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
     5761 agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc
     5821 catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt
     5881 cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
     5941 ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag
     6001 ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
     6061 gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc
     6121 ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc
     6181 acgatagccg cgctgcctcg tccttcagtt cattcagggc accggacagg tcggtcttga
     6241 caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga
     6301 ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
     6361 cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc
     6421 ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg
     6481 gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata
     6541 aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg
     6601 cgcttgcgtt ttccctt
//
