LOCUS       sequence-426128-        5981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal GST tag and TEV cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  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 5981)
  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-GST-TEV-GG
FEATURES             Location/Qualifiers
     source          1..5981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..59
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        40..58
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    59..83
                     /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             98..120
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             128..781
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             797..817
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             885..902
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(955..973)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      969..1016
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1053..1508
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1140..1159)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1350..1371
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1600..2415)
                     /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(1600..2415)
                     /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     2323..2342
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2407..2426
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2537..3125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3026..3045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3279..3296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3311..3450
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3376..3395)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3536..3558
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3552..3743)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4552..5634)
                     /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     5596..5615
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5635..5712)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5918..5937
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
       61 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
      121 tatacatatg tcaccgatat tgggctattg gaaaattaag ggtttggtgc agcctacccg
      181 cttgcttttg gaatacctgg aagagaagta tgaggaacac ttatacgaaa gagacgaggg
      241 cgacaagtgg cgaaacaaga agttcgagtt aggcctcgaa tttcctaatc tgccttacta
      301 tattgatggt gacgtcaaac tgacacagag catggcgatt attcgttata ttgccgataa
      361 gcataacatg cttggtggat gcccgaagga gcgcgcggag atatctatgt tagaaggagc
      421 agtgttagac atccggtacg gggttagccg tatagcgtat agcaaagatt ttgagactct
      481 gaaagtggac tttctgtcca agctgcccga aatgctgaag atgttcgaag atcgcttgtg
      541 ccataagaca tacttgaacg gcgatcacgt aacacatcct gactttatgc tctatgacgc
      601 cctcgacgta gttctctaca tggatcctat gtgtcttgac gcatttccga agttagtgtg
      661 ctttaagaag cgcattgagg ctatcccgca aatagataag tacctgaagt caagcaagta
      721 catcgcttgg cctctgcaag gatggcaagc aactttcggt ggcggtgacc atcccccaaa
      781 gagtggagga ggttcagaga acctgtactt ccaatccatg agagaccgcg gataattcgg
      841 gtctcactcg agcacccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
      901 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
      961 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1021 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1081 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1141 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1201 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1261 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1321 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1381 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1441 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1501 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1561 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     1621 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1681 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1741 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     1801 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1861 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1921 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     1981 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2041 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     2101 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     2161 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     2221 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     2281 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2341 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     2401 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2461 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2521 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2581 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2641 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2701 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2761 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2821 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2881 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2941 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3001 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3061 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3121 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3181 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3241 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3301 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3361 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3421 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3481 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3541 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3601 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3661 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3721 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3781 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3841 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3901 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3961 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4021 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4081 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4141 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4201 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4261 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4321 aggccgatca tcgtcgcgct ccagcgaaag tggtcctcgc cgaaaatgac ccagagcgct
     4381 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4441 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4501 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4561 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4621 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4681 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4741 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4801 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     4861 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4921 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4981 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5041 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5101 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5161 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5221 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5281 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5341 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5401 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5461 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5521 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5581 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5641 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5701 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5761 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5821 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5881 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5941 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat g
//
