LOCUS       sequence-381310-        5499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  use to create N-terminal PelB and C-terminal Twin-Strep-tagged 
            fusion proteins for periplasmic translocation via PelB signal 
            sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5499)
  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 5499)
  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-CTwinStrep
FEATURES             Location/Qualifiers
     source          1..5499
                     /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      2982..3513
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(3640..3726)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        3743..3820
                     /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             3821..4903
                     /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(3840..3859)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5135..5140,5141..5157,5158..5164)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5172..5188
                     /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     5246..5311
                     /codon_start=1
                     /gene="pelB (fragment)"
                     /product="leader peptide for secretion"
                     /label=pelB signal sequence
                     /translation="MKYLLPTAAAGLLLLAAQPAMA"
     CDS             join(5357..5362,5363..5386,5387..5422,5423..5446)
                     /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 attgtccttt
     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 aggcccgcgt tagcgggccg ggagggttcg
     3001 agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt
     3061 aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc
     3121 gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa
     3181 tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc
     3241 ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag
     3301 gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga
     3361 ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc
     3421 gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa
     3481 gttttccgcg aggtatccac aacgccggcg gccctacatg gctctgctgt agtgagtggg
     3541 ttgcgctccg gcagcggtcc tgatcccccg cagaaaaaaa ggatctcaag aagatccttt
     3601 gatcttttct acggcgcgcc cagctgtcta gggcggcgga tttgtcctac tcaggagagc
     3661 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     3721 tatttgatgc ctttaattaa ttgacaccat cgaatggtgc aaaacctttc gcggtatggc
     3781 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     3841 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     3901 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     3961 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     4021 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     4081 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     4141 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     4201 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     4261 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     4321 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     4381 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     4441 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     4501 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     4561 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     4621 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     4681 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     4741 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     4801 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4861 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4921 taagttagcg cgaattgatc tggtttgaca gcttatcatc gactgcacgg tgcaccaatg
     4981 cttctggcgt caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg
     5041 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     5101 ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc cggctcgtat
     5161 aatgtgtgga attgtgagcg gataacaatt tcacacccta ggccgcggcc gcgcgaattg
     5221 ttaactttaa gaaggagata tacatatgaa gtacctgctg ccgaccgccg ccgccggcct
     5281 gctgctgctg gccgcccagc cggccatggc cgatccgaat tcgagctccg tcgactctag
     5341 agcggccgca ctcgagagcg cttggagcca cccgcagttc gagaaaggtg gaggttccgg
     5401 aggtggatcg ggaggttcgg cgtggagcca cccgcagttc gaaaaataat gatgaaagct
     5461 tgcggccgcg tcgtgactgg gaaaaccctg gcgactagt
//
