LOCUS       sequence-426307-        5513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 Twin Strep 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 5513)
  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 5513)
  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-NHSTIIT-AVITAG-GG
FEATURES             Location/Qualifiers
     source          1..5513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..62
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        43..61
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    62..86
                     /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             101..123
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             134..151
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(167..190,191..226,227..250)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             275..295
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             420..437
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(490..508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      504..551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      588..1043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(675..694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     885..906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1135..1950)
                     /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(1135..1950)
                     /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     1858..1877
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1942..1961
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2072..2660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2561..2580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2814..2831
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2846..2985
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2911..2930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3071..3093
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3087..3278)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4087..5169)
                     /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     5131..5150
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5170..5247)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5453..5472
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag
       61 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
      121 agatatacat atgcaccatc atcatcatca ttcttctggt gcgagctgga gtcacccaca
      181 attcgagaaa ggtggtggct ctggtggcgg ttcaggcggc tccgcctggt cccatccaca
      241 gtttgaaaag ggttctggtg tagatctggg taccgagaac ctgtacttcc aatccggatc
      301 tctgaacgat atttttgaag cgcagaaaat tgaatggcat gaatctggtt ccatgagaga
      361 ccgcggataa ttcgggtctc actcgagcac ccgtcgacaa gcttgcggcc gcactcgagc
      421 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      481 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      541 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
      601 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      661 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      721 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
      781 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
      841 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
      901 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
      961 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1021 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1081 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1141 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1201 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1261 ggcaagatcc tggtatcggt ctgctattcc gactcgtcca acatcaatac aacctattaa
     1321 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1381 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     1441 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     1501 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     1561 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     1621 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     1681 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     1741 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     1801 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     1861 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     1921 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     1981 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2041 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2101 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2161 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2221 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2281 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2341 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     2401 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     2461 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     2521 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     2581 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     2641 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     2701 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     2761 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     2821 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     2881 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     2941 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3001 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3061 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3121 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3181 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3241 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3301 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3361 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     3421 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     3481 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     3541 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     3601 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     3661 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     3721 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     3781 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     3841 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagtggtc ctcgccgaaa
     3901 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     3961 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4021 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4141 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4201 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4261 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4321 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4381 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     4441 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     4501 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     4561 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     4621 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     4681 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     4741 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     4801 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     4861 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     4921 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     4981 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5041 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5101 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5161 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5221 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5281 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5341 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     5401 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     5461 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acg
//
