LOCUS       sequence-426137-        5456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 Twin Strep tag and TEV 
            cleavage .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5456)
  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 5456)
  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-GG
FEATURES             Location/Qualifiers
     source          1..5456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             121..138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(154..177,178..213,214..237)
                     /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             262..282
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             350..367
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(420..438)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      434..481
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      518..973
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(605..624)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     815..836
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1065..1880)
                     /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(1065..1880)
                     /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     1788..1807
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1872..1891
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2002..2590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2491..2510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2744..2761
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2776..2915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2841..2860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3001..3023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3017..3208)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4017..5099)
                     /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     5061..5080
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5100..5177)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5383..5402
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      121 caccatcatc atcatcattc ttctggtgcg agctggagtc acccacaatt cgagaaaggt
      181 ggtggctctg gtggcggttc aggcggctcc gcctggtccc atccacagtt tgaaaagggt
      241 tctggtgtag atctgggtac cgagaacctg tacttccaat ccatgagaga ccgcggataa
      301 ttcgggtctc actcgagcac ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
      361 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      421 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
      481 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
      541 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      601 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      661 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
      721 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
      781 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
      841 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
      901 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
      961 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1021 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     1081 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1141 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1201 tggtatcggt ctgctattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1261 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1321 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1381 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1441 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1501 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1561 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1621 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     1681 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     1741 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     1801 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     1861 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     1921 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     1981 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2041 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2101 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2161 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2221 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2281 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2341 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2401 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2461 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2521 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2581 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2641 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     2701 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     2761 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2821 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     2881 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     2941 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3001 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3061 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3121 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3181 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3241 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3301 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3361 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3421 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3481 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3541 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     3601 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     3661 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     3721 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     3781 gcgacaggcc gatcatcgtc gcgctccagc gaaagtggtc ctcgccgaaa atgacccaga
     3841 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     3901 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     3961 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4021 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4081 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4141 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4201 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4261 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4321 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4381 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4441 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4501 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4561 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4621 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4681 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4741 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4801 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     4861 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     4921 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     4981 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5041 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5101 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5161 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5221 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5281 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5341 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5401 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgt
//
