LOCUS       sequence-426306-        6833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 NUSA tag and TEV 
            cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6833)
  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 6833)
  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-NUSA-TEV-GG
FEATURES             Location/Qualifiers
     source          1..6833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        39..57
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    58..82
                     /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             97..119
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             130..147
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1648..1668
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1736..1753
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1806..1824)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1820..1867
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1904..2359
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1991..2010)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2201..2222
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2451..3266)
                     /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(2451..3266)
                     /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     3174..3193
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3258..3277
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3388..3976
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3877..3896
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4130..4147
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4162..4301
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4227..4246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4387..4409
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4403..4594)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5403..6485)
                     /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     6447..6466
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6486..6563)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6769..6788
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
       61 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
      121 atacatatgc atcaccacca tcaccacggc agcggcaaca aggagatcct ggcggttgtt
      181 gaagccgtga gcaacgaaaa ggccctgcct cgcgagaaga tcttcgaagc ccttgagtcc
      241 gcacttgcga cggctacaaa gaagaagtac gagcaagaga ttgacgtgag agtccaaata
      301 gaccgtaaaa gcggagactt cgatactttc cgtcgctggt tagttgttga tgaagtgaca
      361 cagccgacaa aggagattac tttagaagca gcccgttatg aagacgagtc tctgaacctc
      421 ggcgactacg ttgaggatca aatcgaaagt gtgaccttcg acagaatcac gacccaaacg
      481 gcaaaacaag taatcgtaca gaaggtacgt gaagccgaaa gagcaatggt ggttgatcaa
      541 ttccgagaac atgagggcga gataatcacg ggtgttgtaa agaaggtaaa cagagacaac
      601 attagtctgg atcttggaaa caatgctgag gctgtcattc tgcgagaaga catgctccct
      661 cgtgaaaatt ttcggccggg tgacagagta cggggagtgc tctactctgt tcgccccgaa
      721 gcgagaggag cacaattgtt tgttacccgt agtaagcccg agatgttaat cgaattgttt
      781 cggatagagg tcccagagat cggagaggaa gtcattgaga tcaaggcggc agcacgcgac
      841 cctgggagta gagcaaagat cgcggttaag acgaacgata aacgcataga cccagtaggt
      901 gcttgcgttg gtatgcgggg tgctcgcgtg caagctgtat cgacggaatt aggtggagaa
      961 cggatcgata tcgtattatg ggacgacaac ccagcgcaat ttgttattaa cgcgatggcg
     1021 cccgctgatg ttgcttcaat agtcgttgac gaagacaaac atacaatgga catagctgtt
     1081 gaggccggca atcttgccca agccatcggc cgcaacggtc agaatgttcg gcttgctagt
     1141 caattgtctg gttgggagtt gaatgtaatg actgttgacg accttcaggc gaagcaccaa
     1201 gcagaggcac atgcggcaat cgatacgttc actaagtact tggacataga cgaggacttt
     1261 gccactgttc ttgtggaaga gggattcagc acccttgaag aactggccta cgtgcctatg
     1321 aaggagttgc ttgaaataga ggggttagac gaaccaaccg tagaggcgct gcgagaacga
     1381 gcgaagaacg cgctggccac aatcgcgcaa gcccaagagg aatccttagg agataataag
     1441 cctgctgacg atttattaaa cttggaaggt gtagaccgtg accttgcatt caaattggca
     1501 gctcggggtg tttgtacgtt ggaagacctg gccgaacagg gcattgatga tcttgcagac
     1561 attgaaggac tcacagacga aaaggctgga gcccttatca tggcagcccg gaacatttgt
     1621 tggtttgggg acgaggcggg catcgaggag aacctgtact tccaatccat gagagaccgc
     1681 ggataattcg ggtctcactc gagcacccgt cgacaagctt gcggccgcac tcgagcacca
     1741 ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc
     1801 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
     1861 ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc
     1921 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1981 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2041 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2101 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2161 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2221 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2281 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2341 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2401 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac
     2461 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     2521 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2581 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2641 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2701 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2761 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     2821 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     2881 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     2941 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     3001 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     3061 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     3121 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     3181 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa
     3241 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3301 agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3361 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3421 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3481 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3541 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3601 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3661 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3721 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3781 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3841 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3901 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3961 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4021 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4081 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4141 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4201 ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     4261 tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac
     4321 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4381 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     4441 agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat
     4501 ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg
     4561 ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc
     4621 tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg
     4681 atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc
     4741 ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag
     4801 gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg
     4861 gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg
     4921 ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg
     4981 attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga
     5041 gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga
     5101 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     5161 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gtggtcctcg ccgaaaatga
     5221 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5281 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5341 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5401 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5461 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     5521 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     5581 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     5641 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     5701 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     5761 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     5821 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     5881 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     5941 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     6001 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     6061 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     6121 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     6181 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     6241 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     6301 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     6361 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     6421 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     6481 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     6541 gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat
     6601 taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc
     6661 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
     6721 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
     6781 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgc
//
