LOCUS       sequence-426134-        5684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 Thioredoxin tag and TEV 
            cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5684)
  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 5684)
  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-HIS6-THRX-TEV-GG
FEATURES             Location/Qualifiers
     source          1..5684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        46..64
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    65..89
                     /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             104..126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             140..157
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             506..526
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             594..611
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(664..682)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      678..725
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      762..1217
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(849..868)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1059..1080
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1309..2124)
                     /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(1309..2124)
                     /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     2032..2051
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2116..2135
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2246..2834
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2735..2754
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2988..3005
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3020..3159
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3085..3104)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3245..3267
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3261..3452)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4261..5343)
                     /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     5305..5324
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5344..5421)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5627..5646
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
       61 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
      121 aggagatata catatggggc atcatcatca ccaccacggt tcgggtgata aaatcatcca
      181 cctgacagac gactccttcg acacagacgt tctcaaagcg gacggagcta ttctcgttga
      241 cttttgggct gagtggtgcg ggccgtgcaa gatgatagcg ccaattctcg acgagatagc
      301 agatgagtat cagggtaagt taaccgtggc taagttgaat atagaccaga accctggcac
      361 agcccctaag tacggcatcc gagggatacc aaccttatta ctcttcaaga atggtgaggt
      421 ggcagcgacc aaggtcgggg cgttgtccaa ggggcagttg aaggaatttc tggatgcgaa
      481 ccttgccggc ggatcgggcg gatcggagaa tttgtacttc caaagcatga gagaccgcgg
      541 ataattcggg tctcactcga gcacccgtcg acaagcttgc ggccgcactc gagcaccacc
      601 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
      661 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
      721 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
      781 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      841 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      901 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      961 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1021 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1081 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     1141 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     1201 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1261 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     1321 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     1381 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1441 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1501 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1561 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     1621 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     1681 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     1741 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     1801 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     1861 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     1921 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     1981 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     2041 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     2101 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     2161 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2221 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2281 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2341 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2401 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2461 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2521 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2581 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2641 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2701 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2761 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2821 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2881 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2941 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3001 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3061 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3121 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     3181 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     3241 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     3301 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     3361 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     3421 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     3481 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     3541 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     3601 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     3661 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     3721 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     3781 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     3841 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     3901 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     3961 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     4021 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagt ggtcctcgcc gaaaatgacc
     4081 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     4141 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     4201 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     4261 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4321 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     4381 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     4441 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     4501 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     4561 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     4621 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     4681 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     4741 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     4801 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     4861 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     4921 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     4981 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     5041 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     5101 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     5161 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     5221 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     5281 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     5341 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     5401 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     5461 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     5521 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     5581 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     5641 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggcc
//
