LOCUS       sequence-64651-e        5433 bp    DNA     circular SYN 14-APR-2025
DEFINITION  use to create N-terminal Twin-Strep-tag fusion proteins or dual 
            tagged fusion with N-terminal Twin-Strep-tag and C-terminal 6x 
            His-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5433)
  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 5433)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pTD-NTwinStrep_Km
FEATURES             Location/Qualifiers
     source          1..5433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(48..65)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(join(162..185,186..221,222..245,246..251))
                     /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"
     protein_bind    305..321
                     /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)."
     promoter        complement(join(329..335,336..352,353..358))
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             complement(590..1672)
                     /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     1634..1653
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1673..1750)
                     /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."
     terminator      1767..1853
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      1980..2511
                     /label=oriV
                     /note="incP origin of replication"
     CDS             2904..4052
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     oriT            4113..4221
                     /note="incP origin of transfer"
     CDS             complement(4388..5203)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5195..5214
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(5335..5429)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 actagtcgcc agggttttcc cagtcacgac gcggccgcaa gcttttagtg atggtgatgg
       61 tgatgcgatc ctctcaggcc tgagaccatg gtccccctgc aggtcgacct cgagggatcc
      121 ccgggtaccg agctcgaatt cgggaccgcg gtctcggcgc ctttttcgaa ctgcgggtgg
      181 ctccacgccg aacctcccga tccacctccg gaacctccac ctttctcgaa ctgcgggtgg
      241 ctccaagcgc tagccatttg tatatctcct tccaattcgc gcggccgcgg cctagggtgt
      301 gaaattgtta tccgctcaca attccacaca ttatacgagc cggatgatta attgtcaaca
      361 gctcatttca gaatatttgc cagaaccgtt atgatgtcgg cgcaaaaaac attatccaga
      421 acgggagtgc gccttgagcg acacgaatta tgcagtgatt tacgacctgc acagccatac
      481 cacagcttcc gatggctgcc tgacgccaga agcattggtg caccgtgcag tcgatgataa
      541 gctgtcaaac cagatcaatt cgcgctaact tacattaatt gcgttgcgct cactgcccgc
      601 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      661 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
      721 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
      781 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
      841 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
      901 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
      961 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     1021 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     1081 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     1141 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     1201 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     1261 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     1321 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     1381 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     1441 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     1501 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     1561 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     1621 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     1681 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     1741 cgatggtgtc aattaattaa aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     1801 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc
     1861 cctagacagc tgggcgcgcc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1921 tgcgggggat caggaccgct gccggagcgc aacccactca ctacagcaga gccatgtagg
     1981 gccgccggcg ttgtggatac ctcgcggaaa acttggccct cactgacaga tgaggggcgg
     2041 acgttgacac ttgaggggcc gactcacccg gcgcggcgtt gacagatgag gggcaggctc
     2101 gatttcggcc ggcgacgtgg agctggccag cctcgcaaat cggcgaaaac gcctgatttt
     2161 acgcgagttt cccacagatg atgtggacaa gcctggggat aagtgccctg cggtattgac
     2221 acttgagggg cgcgactact gacagatgag gggcgcgatc cttgacactt gaggggcaga
     2281 gtgctgacag atgaggggcg cacctattga catttgaggg gctgtccaca ggcagaaaat
     2341 ccagcatttg caagggtttc cgcccgtttt tcggccaccg ctaacctgtc ttttaacctg
     2401 cttttaaacc aatatttata aaccttgttt ttaaccaggg ctgcgccctg tgcgcgtgac
     2461 cgcgcacgcc gaaggggggt gccccccctt ctcgaaccct cccggcccgc taacgcgggc
     2521 ctcccatccc cccaggggct gcgcccctcg gccgcgaacg gcctcacccc aaaaatggca
     2581 gccacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
     2641 gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
     2701 acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     2761 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     2821 ccaaggatct gatggcgcag gggatcaaga tcgacggatc gatccgggga attaattccg
     2881 gggcaatccc gcaaggaggg tgaatgaatc ggacgtttga ccggaaggca tacaggcaag
     2941 aactgatcga cgcggggttt tccgccgagg atgccgaaac catcgcaagc cgcaccgtca
     3001 tgcgtgcgcc ccgcgaaacc ttccagtccg tcggctcgat agtccagcaa gctacggcca
     3061 agatcgagcg cgacagcgtg caactggctc cccctgccct gcccgcgcca tcggccgccg
     3121 tggagcgttc gcgtcgtctc gaacaggagg cggcaggttt ggcgaagtcg atgaccatcg
     3181 acacgcgagg aactatgacg accaagaagc gaaaaaccgc cggcgaggac ctggcaaaac
     3241 aggtcagcga agccaagcag gccgcgttgc tgaaacacac gaagcagcag atcaaggaaa
     3301 tgcagctttc cttgttcgat attgcgccgt ggccggacac gatgcgagcg atgccaaacg
     3361 acacggcccg ctctgccctg ttcaccacgc gcaacaagaa aatcccgcgc gaggcgctgc
     3421 aaaacaaggt cattttccac gtcaacaagg acgtgaagat cacctacacc ggcgtcgagc
     3481 tgcgggccga cgatgacgaa ctggtgtggc agcaggtgtt ggagtacgcg aagcgcaccc
     3541 ctatcggcga gccgatcacc ttcacgttct acgagctttg ccaggacctg ggctggtcga
     3601 tcaatggccg gtattacacg aaggccgagg aatgcctgtc gcgcctacag gcgacggcga
     3661 tgggcttcac gtccgaccgc gttgggcacc tggaatcggt gtcgctgctg caccgcttcc
     3721 gcgtcctgga ccgtggcaag aaaacgtccc gttgccaggt cctgatcgac gaggaaatcg
     3781 tcgtgctgtt tgctggcgac cactacacga aattcatatg ggagaagtac cgcaagctgt
     3841 cgccgacggc ccgacggatg ttcgactatt tcagctcgca ccgggagccg tacccgctca
     3901 agctggaaac cttccgcctc atgtgcggat cggattccac ccgcgtgaag aagtggcgcg
     3961 agcaggtcgg cgaagcctgc gaagagttgc gaggcagcgg cctggtggaa cacgcctggg
     4021 tcaatgatga cctggtgcat tgcaaacgct agggccttgt ggggtcagtt ccggctgggg
     4081 gttcagcagc cacctgcatc gcggccggcc tacggccagc ctcgcagagc aggattcccg
     4141 ttgagcaccg ccaggtgcga ataagggaca gtgaagaagg aacacccgct cgcgggtggg
     4201 cctacttcac ctatcctgcc cggctgacgc cgttggatac accaaggaaa gtctacacga
     4261 accctttggc aaaatcctgt atatcgtgcg aaaaaggatg gatataccga aaaaatcgct
     4321 ataatgaccc cgaagcaggg ttatgcagcg gaaaaggaca acgcgcggac cgcggtccaa
     4381 ttaattatta gaaaaattca tccagcatca gatgaaattg cagtttgttc atatccggat
     4441 tatcaatgcc atatttctga aacagacgtt tttgcaggct cgggctaaat tcgcccaggc
     4501 agttccacag aatggccaga tcctgataac gatccgcaat gcccacacgg cccacatcaa
     4561 tgcagccaat cagtttgcct tcatcgaaaa tcaggttatc caggctaaaa tcgccgtggg
     4621 tcaccacgct atccgggcta aacggcagca gtttatgcat ttctttccac acctgttcca
     4681 ccggccagcc gttacgttca tcatcaaaat cgctcgcatc caccaggccg ttgttcatac
     4741 ggctctgcgc ctgggccaga cgaaacacac gatcgctgtt aaacgggcag ttgcacaccg
     4801 gaatgctatg cagacgacgc agaaacacgg ccagcgcatc cacaatgttt tcgccgctat
     4861 ccggatattc ttccagcacc tgaaacgcgg ttttgcccgg aatcgcggtg gtcagcagcc
     4921 acgcatcatc cggggtgcga ataaaatgtt taatggtcgg cagcggcata aattcggtca
     4981 gccagttcag acgcaccatt tcatcggtca catcgttcgc cacgctgcct ttgccatgtt
     5041 tcagaaacag ttccggcgca tccggtttgc catacagacg ataaatggtc gcgccgctct
     5101 gacccacgtt atcacgcgcc catttatagc catacagatc cgcatccatg ttgctgttca
     5161 gacgcggacg gctacagctc gtttcacgct gaatatggct cataacaccc cttgtattac
     5221 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
     5281 aacatcagag attttgagac acaaatttaa atcgtaatta ttggggaccc ctggattctc
     5341 accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat ggagttctga
     5401 ggtcattact ggatctatca acaggagtcc aag
//
