LOCUS       sequence-426305-        6008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 GST tag and TEV 
            cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6008)
  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 6008)
  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-GST-TEV-GG
FEATURES             Location/Qualifiers
     source          1..6008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..71
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        52..70
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    71..95
                     /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             110..132
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             143..160
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             167..820
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             836..856
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             924..941
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(994..1012)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1008..1055
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1092..1547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1179..1198)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1389..1410
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1639..2454)
                     /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(1639..2454)
                     /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     2362..2381
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2446..2465
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2576..3164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3065..3084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3318..3335
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3350..3489
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3415..3434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3575..3597
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3591..3782)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4591..5673)
                     /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     5635..5654
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5674..5751)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5957..5976
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
       61 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
      121 ttaagaagga gatatacata tgcaccatca ccaccatcac tcatcgatgt ctcccatttt
      181 gggttattgg aagataaagg ggttagtaca accgaccaga ttactgttag agtaccttga
      241 agagaagtat gaagagcatt tatatgagcg cgacgaaggc gataagtggc gcaacaagaa
      301 atttgagtta ggcctcgagt ttccgaacct tccttattat atcgacgggg acgtaaagtt
      361 gacgcaatct atggcaataa ttcggtatat agcagacaag cataacatgt tgggtggctg
      421 ccccaaagag cgtgccgaga tctccatgtt agaaggagcc gtcctggaca ttcgctatgg
      481 tgtgtcgcgc attgcctact caaaagattt cgagacttta aaagttgact ttctgagcaa
      541 gctgccggag atgcttaaga tgttcgaaga tcggctgtgt cacaagacgt acctcaacgg
      601 tgaccacgtt acccacccag actttatgct ttacgatgcg ctggacgttg tgctttacat
      661 ggacccgatg tgtctggacg cgtttccgaa gttggtatgt ttcaagaagc ggattgaggc
      721 tattccccaa atagacaaat accttaagag tagtaagtac atcgcgtggc cacttcaagg
      781 ttggcaagct accttcggcg gcggcgacca tcccccaaag tcgggcggcg gcagcgagaa
      841 cctgtacttc caatccatga gagaccgcgg ataattcggg tctcactcga gcacccgtcg
      901 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
      961 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1021 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1081 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1141 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1201 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1261 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1321 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1381 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1441 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1501 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1561 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1621 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1681 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     1741 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     1801 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1861 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1921 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1981 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     2041 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     2101 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     2161 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     2221 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     2281 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     2341 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     2401 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     2461 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     2521 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2581 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2641 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2701 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2761 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2821 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2881 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2941 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3001 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3061 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3121 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3181 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3241 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3301 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3361 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3421 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3481 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3541 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3601 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3661 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3721 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3781 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3841 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3901 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3961 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4021 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4081 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4141 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4201 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4261 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4321 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4381 cagcgaaagt ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4441 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4501 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4561 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4621 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4681 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4741 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4801 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4861 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4921 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4981 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5041 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5101 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5161 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5221 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5281 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5341 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5401 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5461 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5521 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5581 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5641 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5701 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5761 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5821 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5881 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5941 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6001 cggtgatg
//
