LOCUS       sequence-426310-        6065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 GST tag and TEV cleavage
            non cleavable N-terminal avitag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6065)
  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 6065)
  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-AVITAG-GG
FEATURES             Location/Qualifiers
     source          1..6065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        42..60
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    61..85
                     /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             100..122
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             133..150
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             157..810
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             826..846
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             971..988
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1041..1059)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1055..1102
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1139..1594
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1226..1245)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1436..1457
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1686..2501)
                     /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(1686..2501)
                     /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     2409..2428
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2493..2512
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2623..3211
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3112..3131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3365..3382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3397..3536
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3462..3481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3622..3644
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3638..3829)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4638..5720)
                     /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     5682..5701
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5721..5798)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6004..6023
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
       61 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
      121 gatatacata tgcaccatca ccaccatcac tcatcgatgt ctcccatttt gggttattgg
      181 aagataaagg ggttagtaca accgaccaga ttactgttag agtaccttga agagaagtat
      241 gaagagcatt tatatgagcg cgacgaaggc gataagtggc gcaacaagaa atttgagtta
      301 ggcctcgagt ttccgaacct tccttattat atcgacgggg acgtaaagtt gacgcaatct
      361 atggcaataa ttcggtatat agcagacaag cataacatgt tgggtggctg ccccaaagag
      421 cgtgccgaga tctccatgtt agaaggagcc gtcctggaca ttcgctatgg tgtgtcgcgc
      481 attgcctact caaaagattt cgagacttta aaagttgact ttctgagcaa gctgccggag
      541 atgcttaaga tgttcgaaga tcggctgtgt cacaagacgt acctcaacgg tgaccacgtt
      601 acccacccag actttatgct ttacgatgcg ctggacgttg tgctttacat ggacccgatg
      661 tgtctggacg cgtttccgaa gttggtatgt ttcaagaagc ggattgaggc tattccccaa
      721 atagacaaat accttaagag tagtaagtac atcgcgtggc cacttcaagg ttggcaagct
      781 accttcggcg gcggcgacca tcccccaaag tcgggcggcg gcagcgagaa cctgtacttc
      841 caatccggat ctctgaacga tatttttgaa gcgcagaaaa ttgaatggca tgaatctggt
      901 tccatgagag accgcggata attcgggtct cactcgagca cccgtcgaca agcttgcggc
      961 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
     1021 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1081 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1141 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1201 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1261 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1321 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1381 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1441 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1501 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1561 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     1621 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1681 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1741 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     1801 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     1861 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     1921 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     1981 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2041 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2101 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2161 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2221 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2281 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2341 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2401 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2461 cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2521 tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt
     2581 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2641 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2701 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2761 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2821 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2881 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     2941 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3001 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3061 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3121 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3181 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3241 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3301 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3361 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3421 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3481 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3541 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3601 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3661 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3721 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3781 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3841 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3901 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     3961 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4021 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4081 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4141 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4201 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4261 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     4321 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     4381 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagtggt
     4441 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     4501 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     4561 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4621 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4681 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4741 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4801 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4861 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4921 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4981 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     5041 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     5101 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     5161 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     5221 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5281 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5341 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5401 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5461 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5521 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5581 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5641 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5701 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5761 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5821 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5881 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     5941 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     6001 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     6061 cacga
//
