pBb(RSF1010)1k-GFPuv

LOCUS      pBb(RSF1010)1k-GFPuv               7107 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  IPTG-inducible trc promoter expressing gfpuv on RSF1010 origin.
ACCESSION   .
VERSION     .
KEYWORDS    pBb(RSF1010)1k-GFPuv
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7107)
  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 7107)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7107
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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        1399..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(3207..4046)
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             complement(4557..5528)
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     rep_origin      5635..6029
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     terminator      6052..6146
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6177..6971)
                     /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(6288..6307)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6898..6917
                     /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 tacctaggtc agcctgccgc cttgggccgg gtgatgtcgt
     2401 acttgcccgc cgcgaactcg gttaccgtcc agcccagcgc gaccagctcc ggcaacgcct
     2461 cgcgcacccg ctggcggcgc ttgcgcatgg tcgaaccact ggcctctgac ggccagacat
     2521 agccgcacaa ggtatctatg gaagccttgc cggttttgcc ggggtcgatc cagccacaca
     2581 gccgctggtg cagcaggcgg gcggtttcgc tgtccagcgc ccgcacctcg tccatgctga
     2641 tgcgcacatg ctggccgcca cccatgacgg cctgcgcgat caaggggttc agggccacgt
     2701 acaggcgccc gtccgcctcg tcgctggcgt actccgacag cagccgaaac ccctgccgct
     2761 tgcggccatt ctgggcgatg atggatacct tccaaaggcg ctcgatgcag tcctgtatgt
     2821 gcttgagcgc cccaccacta tcgacctctg ccccgatttc ctttgccagc gcccgatagc
     2881 tacctttgac caccatggca tcagcggtga cggcctccca cttgggttcc aggaacagcc
     2941 ggagctgccg tccgccttcg gtcttgggtt ccgggccaag cactaggcca ttaggcccag
     3001 ccatggccac cagcccttgc aggatgcgca gatcatcagc gcccagcggc tccgggccgc
     3061 tgaactcgat ccgcttgccg tcgccgtagt catacgtcac gtccagcttg ctgcgcttgc
     3121 gctcgccccg cttgagggca cggaacaggc cgggggccag acagtgcgcc gggtcgtgcc
     3181 ggacgtggct gaggctgtgc ttgttcttag gcttcaccac ggggcacccc cttgctcttg
     3241 cgctgcctct ccagcacggc gggcttgagc accccgccgt catgccgcct gaaccaccga
     3301 tcagcgaacg gtgcgccata gttggccttg ctcacaccga agcggacgaa gaaccggcgc
     3361 tggtcgtcgt ccacacccca ttcctcggcc tcggcgctgg tcatgctcga caggtaggac
     3421 tgccagcgga tgttatcgac cagtaccgag ctgccccggc tggcctgctg ctggtcgcct
     3481 gcgcccatca tggccgcgcc cttgctggca tggtgcagga acacgataga gcacccggta
     3541 tcggcggcga tggcctccat gcgaccgatg acctgggcca tggggccgct ggcgttttct
     3601 tcctcgatgt ggaaccggcg cagcgtgtcc agcaccatca ggcggcggcc ctcggcggcg
     3661 cgcttgaggc cgtcgaacca ctccggggcc atgatgttgg gcaggctgcc gatcagcggc
     3721 tggatcagca ggccgtcagc cacggcttgc cgttcctcgg cgctgaggtg cgccccaagg
     3781 gcgtgcaggc ggtgatgaat ggcggtgggc gggtcttcgg cgggcaggta gatcaccggg
     3841 ccggtgggca gttcgcccac ctccagcaga tccggcccgc ctgcaatctg tgcggccagt
     3901 tgcagggcca gcatggattt accggcacca ccgggcgaca ccagcgcccc gaccgtaccg
     3961 gccaccatgt tgggcaaaac gtagtccagc ggtggcggcg ctgctgcgaa cgcctccaga
     4021 atattgatag gcttatgggt agccattgat tgcctccttt gcaggcagtt ggtggttagg
     4081 cgctggcggg gtcactaccc ccgccctgcg ccgctctgag ttcttccagg cactcgcgca
     4141 gcgcctcgta ttcgtcgtcg gtcagccaga acttgcgctg acgcatccct ttggccttca
     4201 tgcgctcggc atatcgcgct tggcgtacag cgtcagggct ggccagcagg tcgccggtct
     4261 gcttgtcctt ttggtctttc atatcagtca ccgagaaact tgccggggcc gaaaggcttg
     4321 tcttcgcgga acaaggacaa ggtgcagccg tcaaggttaa ggctggccat atcagcgact
     4381 gaaaagcggc cagcctcggc cttgtttgac gtataaccaa agccaccggg caaccaatag
     4441 cccttgtcac ttttgatcag gtagaccgac cctgaagcgc ttttttcgta ttccataaaa
     4501 cccccttctg tgcgtgagta ctcatagtat aacaggcgtg agtaccaacg caagcactac
     4561 atgctgaaat ctggcccgcc cctgtccatg cctcgctggc ggggtgccgg tgcccgtgcc
     4621 agctcggccc gcgcaagctg gacgctgggc agacccatga ccttgctgac ggtgcgctcg
     4681 atgtaatccg cttcgtggcc gggcttgcgc tctgccagcg ctgggctggc ctcggccatg
     4741 gccttgccga tttcctcggc actgcggccc cggctggcca gcttctgcgc ggcgataaag
     4801 tcgcacttgc tgaggtcatc accgaagcgc ttgaccagcc cggccatctc gctgcggtac
     4861 tcgtccagcg ccgtgcgccg gtggcggcta agctgccgct cgggcagttc gaggctggcc
     4921 agcctgcggg ccttctcctg ctgccgctgg gcctgctcga tctgctggcc agcctgctgc
     4981 accagcgccg ggccagcggt ggcggtcttg cccttggatt cacgcagcag cacccacggc
     5041 tgataaccgg cgcgggtggt gtgcttgtcc ttgcggttgg tgaagcccgc caagcggcca
     5101 tagtggcggc tgtcggcgct ggccgggtcg gcgtcgtact cgctggccag cgtccgggca
     5161 atctgccccc gaagttcacc gcctgcggcg tcggccacct tgacccatgc ctgatagttc
     5221 ttcgggctgg tttccactac cagggcaggc tcccggccct cggctttcat gtcatccagg
     5281 tcaaactcgc tgaggtcgtc caccagcacc agaccatgcc gctcctgctc ggcgggcctg
     5341 atatacacgt cattgccctg ggcattcatc cgcttgagcc atggcgtgtt ctggagcact
     5401 tcggcggctg accattcccg gttcatcatc tggccggtgg tggcgtccct gacgccgata
     5461 tcgaagcgct cacagcccat ggccttgagc tgtcggccta tggcctgcaa agtcctgtcg
     5521 ttcttcatcg ggccaccaag cgattcccac acattatacg agccggaagc ataaagtgta
     5581 gctagatccg aaggatgagc cgggctgaat gatcgaccga gacaggccct gcggggctgc
     5641 acacgcgccc ccacccttcg ggtaggggga aaggccgcta aagcggctaa aagcgctcca
     5701 gcgtatttct gcggggtttg gtgtggggtt tagcgggctt tgcccgcctt tccccctgcc
     5761 gcgcagcggt ggggcggtgt gtagcctagc gcagcgaata gaccagctat ccggcctctg
     5821 gccgggcata ttgggcaagg gcagcagcgc cccacaaggg cgctgataac cgcgcctagt
     5881 ggattattct tagataatca tggatggatt tttccaacac cccgccagcc cccgcccctg
     5941 ctgggtttgc aggtttgggg gcgtgacagt tattgcaggg gttcgtgaca gttattgcag
     6001 gggggcgtga cagttattgc aggggttcgt gacagttaga ctagtgcttg gattctcacc
     6061 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt
     6121 cattactgga tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag
     6181 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
     6241 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
     6301 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
     6361 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
     6421 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
     6481 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
     6541 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
     6601 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
     6661 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
     6721 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
     6781 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
     6841 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
     6901 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
     6961 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcatga tcccctgcgc
     7021 catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta
     7081 ccagagggcg ccccagctgg caattcc
//