LOCUS       sequence-206266-        5057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pBBR6k-GFPuv with Anderson promoter J23101 expressing lacI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5057)
  AUTHORS   Cook TB, Rand JM, Nurani W, Courtney DK, Liu SA, Pfleger BF
  TITLE     Genetic tools for reliable gene expression and recombineering in 
            Pseudomonas putida.
  JOURNAL   J Ind Microbiol Biotechnol. 2018 Jan 3. pii: 
            10.1007/s10295-017-2001-5. doi: 10.1007/s10295-017-2001-5.
  PUBMED    29299733
REFERENCE   2  (bases 1 to 5057)
  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: "J Ind 
            Microbiol Biotechnol. 2018 Jan 3. pii: 10.1007/s10295-017-2001-5. 
            doi: 10.1007/s10295-017-2001-5."
COMMENT     Sequence Label: pBBR6k-J23101-GFPuv
FEATURES             Location/Qualifiers
     source          1..5057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      612..1383
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(1417..1511)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1542..2336)
                     /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(1653..1672)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2263..2282
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(2521..3603)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3565..3584
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3630..3664)
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     primer_bind     3882..3901
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     protein_bind    3969..3985
                     /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)."
     protein_bind    3992..4008
                     /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)."
     CDS             4076..4792
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4106..4134)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4729..4749
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4825..4896
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4912..4939
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(5006..5057,1..611))
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 tccagaaaac acggctcatc gggcatcggc aggcgctgct gcccgcgccg ttcccattcc
       61 tccgtttcgg tcaaggctgg caggtctggt tccatgcccg gaatgccggg ctggctgggc
      121 ggctcctcgc cggggccggt cggtagttgc tgctcgcccg gatacagggt cgggatgcgg
      181 cgcaggtcgc catgccccaa cagcgattcg tcctggtcgt cgtgatcaac caccacggcg
      241 gcactgaaca ccgacaggcg caactggtcg cggggctggc cccacgccac gcggtcattg
      301 accacgtagg ccgacacggt gccggggccg ttgagcttca cgacggagat ccagcgctcg
      361 gccaccaagt ccttgactgc gtattggacc gtccgcaaag aacgtccgat gagcttggaa
      421 agtgtcttct ggctgaccac cacggcgttc tggtggccca tctgcgccac gaggtgatgc
      481 agcagcattg ccgccgtggg tttcctcgca ataagcccgg cccacgcctc atgcgctttg
      541 cgttccgttt gcacccagtg accgggcttg ttcttggctt gaatgccgat ttctctggac
      601 tgcgtggcca tgcttatctc catgcggtag gggtgccgca cggttgcggc accatgcgca
      661 atcagctgca acttttcggc agcgcgacaa caattatgcg ttgcgtaaaa gtggcagtca
      721 attacagatt ttctttaacc tacgcaatga gctattgcgg ggggtgccgc aatgagctgt
      781 tgcgtacccc ccttttttaa gttgttgatt tttaagtctt tcgcatttcg ccctatatct
      841 agttctttgg tgcccaaaga agggcacccc tgcggggttc ccccacgcct tcggcgcggc
      901 tccccctccg gcaaaaagtg gcccctccgg ggcttgttga tcgactgcgc ggccttcggc
      961 cttgcccaag gtggcgctgc ccccttggaa cccccgcact cgccgccgtg aggctcgggg
     1021 ggcaggcggg cgggcttcgc ccttcgactg cccccactcg cataggcttg ggtcgttcca
     1081 ggcgcgtcaa ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg ccttccactt
     1141 ggtgtccaac cggcaagcga agcgcgcagg ccgcaggccg gaggcttttc cccagagaaa
     1201 attaaaaaaa ttgatggggc aaggccgcag gccgcgcagt tggagccggt gggtatgtgg
     1261 tcgaaggctg ggtagccggt gggcaatccc tgtggtcaag ctcgtgggca ggcgcagcct
     1321 gtccatcagc ttgtccagca gggttgtcca cgggccgagc gaagcgagcc agccggtggc
     1381 cgctcgcggc catcgtccac atatcctagt gcttggattc tcaccaataa aaaacgcccg
     1441 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat
     1501 caacaggagt ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     1561 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     1621 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     1681 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     1741 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     1801 ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     1861 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     1921 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     1981 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     2041 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     2101 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt
     2161 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     2221 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     2281 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     2341 aacgatcctc atcctgtctc ttgatcagat catgatcccc tgcgccatca gatccttggc
     2401 ggcaagaaag ccatccagtt tactttgcag ggcttcccaa ccttaccaga gggcgcccca
     2461 gctggcaatt ccgacgtcgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     2521 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     2581 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     2641 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     2701 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     2761 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atgtccgcac caacgcgcag
     2821 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     2881 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     2941 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     3001 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     3061 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     3121 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     3181 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     3241 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     3301 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     3361 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     3421 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     3481 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     3541 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     3601 cacattcacc accctgaatt gactctcttg ctagcataat acctaggact gagctagctg
     3661 taaattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     3721 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     3781 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     3841 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     3901 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     3961 atcgagaatt gtgagcggat aacaattgac attgtgagcg gataacaaga tactgagcac
     4021 atcagcagga cgcactgacc gaattcaaaa gatcttttaa gaaggagata tacatatgag
     4081 taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
     4141 taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact
     4201 tacccttaaa tttatttgca ctactggaaa actacctgtt ccgtggccaa cacttgtcac
     4261 tactttctct tatggtgttc aatgcttttc ccgttatccg gatcacatga aacggcatga
     4321 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga
     4381 tgacgggaac tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg
     4441 tatcgagtta aaaggtattg attttaaaga agatggaaac attctcggac acaaactgga
     4501 gtacaactat aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa
     4561 agctaacttc aaaattcgcc acaacattga agatggctcc gttcaactag cagaccatta
     4621 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
     4681 cacacaatct gccctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga
     4741 gtttgtaact gctgctggga ttacacatgg catggatgag ctctacaaat aaggatccaa
     4801 actcgagtaa ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4861 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     4921 tcgggtgggc ctttctgcgt ttatacctag gctacagccg atagtctgga acagcgcact
     4981 tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag cgtgacccgt gtcggcggct
     5041 ccaacggctc gccatcg
//
