LOCUS       sequence-206280-        5658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible trc promoter expressing gfpuv on RK2 origin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5658)
  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 5658)
  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: pBb(RK2)1k-GFPuv
FEATURES             Location/Qualifiers
     source          1..5658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..84
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             85..1167
                     /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     complement(104..123)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1399..1404,1405..1421,1422..1428)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1436..1452
                     /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             1493..2209
                     /codon_start=1
                     /product="GFP variant optimized for excitation by UV light"
                     /label=GFPuv
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1523..1551)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2146..2166
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2242..2313
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2329..2356
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2419..3567)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      3960..4491
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(4603..4697)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4728..5522)
                     /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(4839..4858)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5449..5468
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gacgtcgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
       61 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
      121 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
      181 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
      241 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
      301 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
      361 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
      421 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
      481 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
      541 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
      601 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
      661 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
      721 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
      781 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
      841 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
      901 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
      961 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     1021 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     1081 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     1141 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt
     1201 gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca
     1261 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
     1321 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
     1381 atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg
     1441 agcggataac aatttcagaa ttcaaaagat cttttaagaa ggagatatac atatgagtaa
     1501 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
     1561 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
     1621 ccttaaattt atttgcacta ctggaaaact acctgttccg tggccaacac ttgtcactac
     1681 tttctcttat ggtgttcaat gcttttcccg ttatccggat cacatgaaac ggcatgactt
     1741 tttcaagagt gccatgcccg aaggttatgt acaggaacgc actatatctt tcaaagatga
     1801 cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat
     1861 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt ctcggacaca aactggagta
     1921 caactataac tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc
     1981 taacttcaaa attcgccaca acattgaaga tggctccgtt caactagcag accattatca
     2041 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
     2101 acaatctgcc ctttcgaaag atcccaacga aaagcgtgac cacatggtcc ttcttgagtt
     2161 tgtaactgct gctgggatta cacatggcat ggatgagctc tacaaataag gatccaaact
     2221 cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     2281 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
     2341 ggtgggcctt tctgcgttta tacctagggc gatgcaggtg gctgctgaac ccccagccgg
     2401 aactgacccc acaaggccct agcgtttgca atgcaccagg tcatcattga cccaggcgtg
     2461 ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg ccgacctgct cgcgccactt
     2521 cttcacgcgg gtggaatccg atccgcacat gaggcggaag gtttccagct tgagcgggta
     2581 cggctcccgg tgcgagctga aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg
     2641 gtacttctcc catatgaatt tcgtgtagtg gtcgccagca aacagcacga cgatttcctc
     2701 gtcgatcagg acctggcaac gggacgtttt cttgccacgg tccaggacgc ggaagcggtg
     2761 cagcagcgac accgattcca ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc
     2821 ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac cggccattga tcgaccagcc
     2881 caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt
     2941 cgcgtactcc aacacctgct gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc
     3001 ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg accttgtttt gcagcgcctc
     3061 gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat
     3121 cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag gaaagctgca tttccttgat
     3181 ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggct tcgctgacct gttttgccag
     3241 gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat
     3301 cgacttcgcc aaacctgccg cctcctgttc gagacgacgc gaacgctcca cggcggccga
     3361 tggcgcgggc agggcagggg gagccagttg cacgctgtcg cgctcgatct tggccgtagc
     3421 ttgctggact atcgagccga cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg
     3481 gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta
     3541 tgccttccgg tcaaacgtcc gattcattca ccctccttgc gggattgccc cggaattaat
     3601 tccccggatc gatccgtcga tcttgatccc ctgcgccatc agatccttgg cggcaagaaa
     3661 gccatccagt ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat
     3721 tccggttcgc ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg
     3781 caagctacct gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg
     3841 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtggc tgccattttt
     3901 ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg gggatgggag gcccgcgtta
     3961 gcgggccggg agggttcgag aagggggggc accccccttc ggcgtgcgcg gtcacgcgca
     4021 cagggcgcag ccctggttaa aaacaaggtt tataaatatt ggtttaaaag caggttaaaa
     4081 gacaggttag cggtggccga aaaacgggcg gaaacccttg caaatgctgg attttctgcc
     4141 tgtggacagc ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac tctgcccctc
     4201 aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt gtcaataccg
     4261 cagggcactt atccccaggc ttgtccacat catctgtggg aaactcgcgt aaaatcaggc
     4321 gttttcgccg atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc gagcctgccc
     4381 ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt ccgcccctca
     4441 tctgtcagtg agggccaagt tttccgcgag gtatccacaa cgccggcggc cctacatggc
     4501 tctgctgtag tgagtgggtt gcgctccggc agcggtcctg atcccccgca gaaaaaaagg
     4561 atctcaagaa gatcctttga tcttttctac actagtgctt ggattctcac caataaaaaa
     4621 cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg
     4681 atctatcaac aggagtccaa gcgagctctc gaaccccaga gtcccgctca gaagaactcg
     4741 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     4801 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     4861 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     4921 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     4981 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     5041 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     5101 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     5161 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     5221 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     5281 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     5341 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     5401 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     5461 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     5521 atgcgaaacg atcctcatcc tgtctcttga tcagatcatg atcccctgcg ccatcagatc
     5581 cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc
     5641 gccccagctg gcaattcc
//
