LOCUS       sequence-64657-e        6200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses P.putida codon optimized PelB-sfYFP-Twin-Strep fusion 
            protein for periplasmic translocation via PelB signal sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6200)
  AUTHORS   Dammeyer T, Timmis KN, Tinnefeld P
  TITLE     Broad host range vectors for expression of proteins with (Twin-) 
            Strep-tag, His-tag and engineered, export optimized yellow 
            fluorescent protein.
  JOURNAL   Microb Cell Fact. 2013 May 20;12(1):49.
  PUBMED    23687945
REFERENCE   2  (bases 1 to 6200)
  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: "Microb Cell
            Fact."; date: "2013-05-20"; volume: "12(1)"; pages: "49"
COMMENT     Sequence Label: pTDpelB-C_sfYFPTwinStrep
FEATURES             Location/Qualifiers
     source          1..6200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      5..99
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             335..1126
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     oriT            1272..1380
                     /note="incP origin of transfer"
     CDS             complement(1441..2589)
                     /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      2981..3512
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(3639..3725)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        3742..3819
                     /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             3820..4902
                     /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(3839..3858)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5134..5139,5140..5156,5157..5163)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5171..5187
                     /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)."
     sig_peptide     5245..5310
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             join(6058..6063,6064..6087,6088..6123,6124..6147)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
ORIGIN
        1 cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga
       61 acgctcggtt gccgccgggc gttttttatt ggtgagaatc caggggtccc caataattac
      121 gatttacgta tttaaatgaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt
      181 tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca
      241 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca
      301 gcagggcagt cgccctaaaa caaagttaaa catcatgagg gaagcggtga tcgccgaagt
      361 atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct
      421 ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga
      481 tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga
      541 ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac
      601 cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt
      661 tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat
      721 tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc
      781 ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac
      841 cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac
      901 gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc
      961 cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca
     1021 ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt
     1081 tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataagaca agggtccttt
     1141 tccgctgcat aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt
     1201 ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa
     1261 cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc
     1321 actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg
     1381 taggccggcc gcgatgcagg tggctgctga acccccagcc ggaactgacc ccacaaggcc
     1441 ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc
     1501 tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc
     1561 cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct
     1621 gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa
     1681 tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca
     1741 acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc
     1801 caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca
     1861 ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc
     1921 gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg
     1981 ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac
     2041 gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt
     2101 gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg
     2161 ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt
     2221 cagcaacgcg gcctgcttgg cttcgctgac ctgttttgcc aggtcctcgc cggcggtttt
     2281 tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc
     2341 cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg
     2401 gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ctatcgagcc
     2461 gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc
     2521 atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt
     2581 ccgattcatt caccctcctt gcgggattgc cccggaatta attccccgga tcgatccgtc
     2641 gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     2701 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
     2761 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc
     2821 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag
     2881 caccgtttct gcggactggc tttctacgtg gctgccattt ttggggtgag gccgttcgcg
     2941 gccgaggggc gcagcccctg gggggatggg gtgccgcgtt agcgggccgg gagggttcga
     3001 gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta
     3061 aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg
     3121 aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat
     3181 gtcaataggt gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc
     3241 cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg
     3301 cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag
     3361 gctggccagc tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg
     3421 ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag
     3481 ttttccgcga ggtatccaca acgccggcgg ccctacatgg ctctgctgta gtgagtgggt
     3541 tgcgctccgg cagcggtcct gatcccccgc agaaaaaaag gatctcaaga agatcctttg
     3601 atcttttcta cggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg
     3661 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     3721 atttgatgcc tttaattaat tgacaccatc gaatggtgca aaacctttcg cggtatggca
     3781 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     3841 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     3901 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     3961 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     4021 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     4081 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     4141 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     4201 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     4261 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     4321 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     4381 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     4441 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     4501 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     4561 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     4621 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     4681 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     4741 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     4801 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     4861 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     4921 aagttagcgc gaattgatct ggtttgacag cttatcatcg actgcacggt gcaccaatgc
     4981 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     5041 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     5101 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc ggctcgtata
     5161 atgtgtggaa ttgtgagcgg ataacaattt cacaccctag gccgcggccg cgcgaattgt
     5221 taactttaag aaggagatat acatatgaag tacctgctgc cgaccgccgc cgccggcctg
     5281 ctgctgctgg ccgcccagcc ggccatggcc gatccgaatt cggtgtcgaa gggcgaagaa
     5341 ctgttcaccg gcgtggtgcc gatcctggtg gaactggacg gcgacgtgaa cggccacaag
     5401 ttccgcgtgt cgggcgaagg cgaaggcgac gccaccaacg gcaagctgac cctgaagttc
     5461 atctgcacca ccggcaagct gccggtgccg tggccgaccc tggtgaccac cctgggctac
     5521 ggcctgcagt gcttcgcccg ctacccggac cacatgaagc agcacgactt cttcaagtcg
     5581 gccatgccgg aaggctacgt gcaggaacgc accatctcgt tcaaggacga cggcacctac
     5641 aagacccgcg ccgaagtgaa gttcgaaggc gacaccctgg tgaaccgcat cgaactgaag
     5701 ggcatcgact tcaaggaaga cggcaacatc ctgggccaca agctggaata caacttcaac
     5761 tcgcacaacg tgtacatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     5821 atccgccaca acgtggaaga cggcggcgtg cagctggccg accactacca gcagaacacc
     5881 ccgatcggcg acggcccggt gctgctgccg gacaaccact acctgtcgta ccagtcgaag
     5941 ctgtcgaagg acccgaacga aaagcgcgac cacatggtgc tgctggaatt tgtgaccgcc
     6001 gccggcatca ccctgggcat ggacgaactg tacaagtcta gagcggccgc actcgagagc
     6061 gcttggagcc acccgcagtt cgagaaaggt ggaggttccg gaggtggatc gggaggttcg
     6121 gcgtggagcc acccgcagtt cgaaaaataa tgatgaaagc ttgcggccgc gtcgtgactg
     6181 ggaaaaccct ggcgactagt
//
