LOCUS       sequence-207207-        4693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible pTRC promoter expressing sfGFP on BBR1-B5 origin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4693)
  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 4693)
  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(B5)8k-sfGFP
FEATURES             Location/Qualifiers
     source          1..4693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..23,24..30)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    38..54
                     /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             join(95..97,98..811)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             95..808
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(125..153)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     748..768
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      844..915
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      931..958
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2240..2334)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2365..3159)
                     /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(2476..2495)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3086..3105
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        3302..3379
                     /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             3380..4462
                     /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(3399..3418)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcag
       61 aattcaaaag atcttttaag aaggagatat acatatgagc aaaggagaag aacttttcac
      121 tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt
      181 ccgtggagag ggtgaaggtg atgctacaaa cggaaaactc acccttaaat ttatttgcac
      241 tactggaaaa ctacctgttc cgtggccaac acttgtcact actctgacct atggtgttca
      301 atgcttttcc cgttatccgg atcacatgaa acggcatgac tttttcaaga gtgccatgcc
      361 cgaaggttat gtacaggaac gcactatatc tttcaaagat gacgggacct acaagacgcg
      421 tgctgaagtc aagtttgaag gtgataccct tgttaatcgt atcgagttaa agggtattga
      481 ttttaaagaa gatggaaaca ttcttggaca caaacttgag tacaacttta actcacacaa
      541 tgtatacatc acggcagaca aacaaaagaa tggaatcaaa gctaacttca aaattcgcca
      601 caacgttgaa gatggttccg ttcaactagc agaccattat caacaaaata ctccaattgg
      661 cgatggccct gtccttttac cagacaacca ttacctgtcg acacaatctg tcctttcgaa
      721 agatcccaac gaaaagcgtg accacatggt ccttcttgag tttgtaactg ctgctgggat
      781 tacacatggc atggatgagc tctacaaata aggatccaaa ctcgagtaag gatctccagg
      841 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
      901 tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt
      961 tatacctagg ctacagccga tagtctggaa cagcgcactt acgggttgct gcgcaaccca
     1021 agtgctaccg gcgcggcagc gtgacccgtg tcggcggctc caacggctcg ccatcgtcca
     1081 gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc cattcctccg
     1141 tttcggtcaa ggctggctgg gcggctcctc gccggggccg gtcggtagtt gctgctcgcc
     1201 cggatacagg gtcgggatgc ggcgcaggtc gccatgcccc aacagcgatt cgtcctggtc
     1261 gtcgtgatca accaccacgg cggcactgaa caccgacagg cgcaactggt cgcggggctg
     1321 gccccacgcc acgcggtcat tgaccacgta ggccaacacg gtgccggggc cgttgagctt
     1381 cacgacggag atccagcgct cggccaccaa gtccttgact gcgtattgga ccgtccgcaa
     1441 agaacgtccg atgagcttgg aaagtgtctt ctggctgacc accacggcgt tctggtggcc
     1501 catctgcgcc acgaggtgat gcagcagcat tgccgccgtg ggtttcctcg caataagccc
     1561 ggcccacgcc tcatgcgctt tgcgttccgt ttgcacccag tgaccgggct tgttcttggc
     1621 ttgaatgccg atttctctgg actgcgtggc catgcttatc tccatgcggt aggggtgccg
     1681 cacggttgcg gcaccatgcg caatcagctg caacttttcg gcagcgcgac aacaattatg
     1741 cgttgcgtaa aagtggcagt caattacaga ttttctttaa cctacgcaat gagctattgc
     1801 ggggggtgcc gcaatgagct gttgcgtacc cccctttttt aagttgttga tttttaagtc
     1861 tttcgcattt cgccctatat ctagttcttt ggtgcccaaa gaagggcacc cctgcggggt
     1921 tcccccacgc cttcggcgcg gctccccctc cggcaaaaag tggcccctcc ggggcttgtt
     1981 gatcgactgc gcggccttcg gccttgccca aggtggcgct gcccccttgg aacccccgca
     2041 ctcgccgccg tgaggctcgg ggggcaggcg ggcgggcttc gcccttcgac tgcccccact
     2101 cgcataggct tgggtcgttc caggcgcgtc aaggccaagc cgctgcgcgg tcgctgcgcg
     2161 agccttgacc cgccttccac ttggtgtcca accggcaagc gaagcgcgca ggccgcaggc
     2221 cggaggcact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
     2281 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg
     2341 agctctcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     2401 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     2461 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     2521 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     2581 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     2641 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     2701 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     2761 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     2821 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     2881 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     2941 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     3001 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     3061 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     3121 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     3181 ctcttgatca gatcatgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca
     3241 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccgacgt
     3301 cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     3361 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     3421 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     3481 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     3541 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     3601 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     3661 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     3721 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     3781 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     3841 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     3901 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     3961 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     4021 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     4081 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     4141 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     4201 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     4261 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     4321 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     4381 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     4441 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagcgc gaattgatct
     4501 ggtttgacag cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat
     4561 cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg
     4621 cgcactcccg ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt
     4681 ctgaaatgag ctg
//
