LOCUS       sequence-426129-        5535 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N-terminal His10 Zbasic tag and TEV 
            cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5535)
  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 5535)
  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-HIS10-ZBASIC-TEV-GG
FEATURES             Location/Qualifiers
     source          1..5535
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..69
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        50..68
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    69..93
                     /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             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             144..173
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             183..353
                     /codon_start=1
                     /product="Z domain of staphylococcal protein A, engineered 
                     to contain multiple positive charges (Hedhammar and Hober, 
                     2007)"
                     /label=Z-basic tag
                     /note="useful for purifying tagged proteins by 
                     cation-exchange chromatography"
                     /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL
                     AEAKKLNDAQPK"
     CDS             363..383
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             451..468
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(521..539)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      535..582
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      619..1074
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(706..725)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     916..937
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1166..1981)
                     /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(1166..1981)
                     /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     1889..1908
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1973..1992
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2103..2691
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2592..2611
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2845..2862
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2877..3016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2942..2961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3102..3124
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4116..5198)
                     /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     5160..5179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5199..5276)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5482..5501
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc
       61 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      121 aagaaggaga tatacatatg gggcatcatc accatcacca tcatcaccac cacagtagcg
      181 gggttgacaa caagttcaac aaggagagac ggcgtgcaag acgcgaaata cgtcacctgc
      241 cgaacctcaa tcgcgaacaa cgccgtgcct tcattcgctc attacgggac gatccttccc
      301 agagcgccaa cctcttagct gaggcaaaga agctgaatga tgcgcaaccc aaaggtagcg
      361 gagaaaattt atattttcag tccatgagag accgaggcta gttcgggtct cactcgagca
      421 cccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
      481 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      541 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      601 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      661 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      721 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      781 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      841 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      901 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
      961 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1021 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1081 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1141 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1201 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1261 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1321 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1381 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     1441 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1501 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     1561 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1621 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1681 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1741 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     1801 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1861 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1921 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     1981 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     2041 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2101 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2161 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2221 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2281 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2341 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2401 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2461 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2521 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2581 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2641 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2701 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2761 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2821 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     2881 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     2941 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3001 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3061 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3121 gaggttttca ccgtcatccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3181 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3241 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3301 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3361 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3421 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3481 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3541 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3601 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     3661 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     3721 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     3781 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     3841 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     3901 cgctccagcg aaagtggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     3961 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4021 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4081 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4141 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4201 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4261 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4321 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4381 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4441 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4501 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4561 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4621 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     4681 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     4741 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     4801 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     4861 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     4921 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     4981 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5041 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5101 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5161 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5221 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5281 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5341 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5401 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5461 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5521 ggcgccggtg atgcc
//
