LOCUS       sequence-426308-        5449 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with C terminal TEV cleavage of His10 and 
            Twin Strep tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5449)
  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 5449)
  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-CTH10STII-GG
FEATURES             Location/Qualifiers
     source          1..5449
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..68
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        49..67
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    68..92
                     /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)."
     CDS             169..189
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             190..219
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(229..252,253..288,289..312)
                     /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             362..379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(432..450)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      446..493
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      530..985
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(617..636)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     827..848
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1077..1892)
                     /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(1077..1892)
                     /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     1800..1819
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1884..1903
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2014..2602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2503..2522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2756..2773
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2788..2927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2853..2872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3013..3035
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3029..3220)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4029..5111)
                     /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     5073..5092
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5112..5189)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5395..5414
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
       61 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacctta
      121 agaaggagat atacacatga gagaccgcgg ataattcggg tctcactcga gaacctctac
      181 ttccaatcgc accatcatca ccaccatcat catcatcacg gttctggttg gagtcaccca
      241 caattcgaga aaggtggtgg ctctggtggc ggttcaggcg gctccgcctg gtcccatcca
      301 cagtttgaaa agtgaggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga
      361 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
      421 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      481 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
      541 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      601 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      661 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      721 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      781 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      841 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      901 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      961 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1021 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     1081 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1141 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1201 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1261 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1321 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1381 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1441 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1501 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1561 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1621 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1681 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1741 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1801 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     1861 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     1921 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1981 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     2041 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     2101 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     2161 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     2221 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     2281 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     2341 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     2401 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     2461 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     2521 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     2581 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2641 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2701 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2761 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     2821 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     2881 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     2941 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3001 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     3061 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     3121 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     3181 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     3241 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     3301 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     3361 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     3421 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     3481 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     3541 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     3601 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     3661 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     3721 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     3781 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     3841 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     3901 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     3961 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4021 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4081 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4141 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4201 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4261 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4321 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4381 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4441 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     4501 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     4561 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     4621 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     4681 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     4741 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     4801 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     4861 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     4921 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     4981 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5041 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5101 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5161 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5221 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5281 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5341 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5401 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccg
//
