LOCUS       sequence-212720-        4692 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 4692)
  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 4692)
  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..4692
                     /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(94..96,97..810)
                     /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             94..807
                     /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(124..152)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     747..767
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      843..914
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      930..957
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2239..2333)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2364..3158)
                     /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(2475..2494)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3085..3104
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        3301..3378
                     /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             3379..4461
                     /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(3398..3417)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcag
       61 aattcaaaag atctttaaga aggagatata catatgagca aaggagaaga acttttcact
      121 ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc
      181 cgtggagagg gtgaaggtga tgctacaaac ggaaaactca cccttaaatt tatttgcact
      241 actggaaaac tacctgttcc gtggccaaca cttgtcacta ctctgaccta tggtgttcaa
      301 tgcttttccc gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc
      361 gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaccta caagacgcgt
      421 gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa gggtattgat
      481 tttaaagaag atggaaacat tcttggacac aaacttgagt acaactttaa ctcacacaat
      541 gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac
      601 aacgttgaag atggttccgt tcaactagca gaccattatc aacaaaatac tccaattggc
      661 gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgt cctttcgaaa
      721 gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt
      781 acacatggca tggatgagct ctacaaataa ggatccaaac tcgagtaagg atctccaggc
      841 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
      901 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
      961 atacctaggc tacagccgat agtctggaac agcgcactta cgggttgctg cgcaacccaa
     1021 gtgctaccgg cgcggcagcg tgacccgtgt cggcggctcc aacggctcgc catcgtccag
     1081 aaaacacggc tcatcgggca tcggcaggcg ctgctgcccg cgccgttccc attcctccgt
     1141 ttcggtcaag gctggctggg cggctcctcg ccggggccgg tcggtagttg ctgctcgccc
     1201 ggatacaggg tcgggatgcg gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg
     1261 tcgtgatcaa ccaccacggc ggcactgaac accgacaggc gcaactggtc gcggggctgg
     1321 ccccacgcca cgcggtcatt gaccacgtag gccaacacgg tgccggggcc gttgagcttc
     1381 acgacggaga tccagcgctc ggccaccaag tccttgactg cgtattggac cgtccgcaaa
     1441 gaacgtccga tgagcttgga aagtgtcttc tggctgacca ccacggcgtt ctggtggccc
     1501 atctgcgcca cgaggtgatg cagcagcatt gccgccgtgg gtttcctcgc aataagcccg
     1561 gcccacgcct catgcgcttt gcgttccgtt tgcacccagt gaccgggctt gttcttggct
     1621 tgaatgccga tttctctgga ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc
     1681 acggttgcgg caccatgcgc aatcagctgc aacttttcgg cagcgcgaca acaattatgc
     1741 gttgcgtaaa agtggcagtc aattacagat tttctttaac ctacgcaatg agctattgcg
     1801 gggggtgccg caatgagctg ttgcgtaccc ccctttttta agttgttgat ttttaagtct
     1861 ttcgcatttc gccctatatc tagttctttg gtgcccaaag aagggcaccc ctgcggggtt
     1921 cccccacgcc ttcggcgcgg ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg
     1981 atcgactgcg cggccttcgg ccttgcccaa ggtggcgctg cccccttgga acccccgcac
     2041 tcgccgccgt gaggctcggg gggcaggcgg gcgggcttcg cccttcgact gcccccactc
     2101 gcataggctt gggtcgttcc aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga
     2161 gccttgaccc gccttccact tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc
     2221 ggaggcacta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct
     2281 gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagcga
     2341 gctctcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg
     2401 cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg
     2461 ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
     2521 cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc
     2581 aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc
     2641 ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg
     2701 acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg
     2761 aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
     2821 actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat
     2881 agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc
     2941 gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac
     3001 aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca
     3061 tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
     3121 gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc
     3181 tcttgatcag atcatgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag
     3241 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccgacgtc
     3301 gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt
     3361 caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt
     3421 gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg
     3481 cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa
     3541 caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac
     3601 gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg
     3661 gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt
     3721 ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt
     3781 gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca
     3841 cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg
     3901 gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg
     3961 cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg
     4021 gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat
     4081 gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg
     4141 cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac
     4201 gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc
     4261 ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag
     4321 ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg
     4381 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     4441 cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagcgcg aattgatctg
     4501 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc
     4561 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc
     4621 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc
     4681 tgaaatgagc tg
//
