LOCUS       sequence-426302-        5519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal TolB SRP pathway signal 
            peptide N terminal His6 Twin Strep tag and TEV cleavage .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5519)
  AUTHORS   Fairhead M, Koekemoer L, Williams E, von Delft F
  TITLE     A selection of Golden Gate vectors to simplify recombinant protein 
            production in Escherichia coli
  JOURNAL   bioRxiv 2024.02.13.579886
REFERENCE   2  (bases 1 to 5519)
  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: "bioRxiv 
            2024.02.13.579886"
COMMENT     Sequence Label: pNIC-TolB-NHSTIIT-GG
FEATURES             Location/Qualifiers
     source          1..5519
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        41..59
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    60..84
                     /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)."
     RBS             99..121
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             195..212
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(228..251,252..287,288..311)
                     /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="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             336..356
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             424..441
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(494..512)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      508..555
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      592..1047
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(679..698)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     889..910
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1139..1954)
                     /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"
     CDS             complement(1139..1954)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /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     1862..1881
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1946..1965
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2076..2664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2565..2584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2818..2835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2850..2989
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2915..2934)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3075..3097
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3091..3282)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4091..5173)
                     /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     5135..5154
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5174..5251)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5457..5476
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
       61 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      121 atatacatat gaaacaggca ttacgcgttg cctttggctt cctgattctg tgggctagcg
      181 tgttgcatgc gatgcaccat catcatcatc attcttctgg tgcgagctgg agtcacccac
      241 aattcgagaa aggtggtggc tctggtggcg gttcaggcgg ctccgcctgg tcccatccac
      301 agtttgaaaa gggttctggt gtagatctgg gtaccgagaa cctgtacttc caatccatga
      361 gagaccgcgg ataattcggg tctcactcga gcacccgtcg acaagcttgc ggccgcactc
      421 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      481 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      541 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      601 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      661 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      721 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      781 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      841 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
      901 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
      961 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1021 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1141 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1201 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1261 ggatggcaag atcctggtat cggtctgcta ttccgactcg tccaacatca atacaaccta
     1321 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1381 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1441 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1501 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1561 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1621 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1681 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1741 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1801 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1861 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1921 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     1981 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2041 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2101 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2161 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2221 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2281 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2341 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2401 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2461 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2521 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2581 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2641 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2701 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2761 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2821 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2881 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2941 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3001 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3061 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3121 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3181 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3241 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3301 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3361 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3421 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3481 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3541 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3601 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3661 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3721 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3781 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3841 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagt ggtcctcgcc
     3901 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     3961 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4021 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4081 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4141 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4201 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4261 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4321 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4381 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4441 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4501 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4561 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4621 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4681 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4741 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4801 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4861 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4921 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4981 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5041 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5101 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5161 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5221 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5281 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5341 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5401 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5461 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgat
//
