LOCUS       sequence-426156-        5509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with C terminal non cleavable AVITAG and TEV 
            cleavage of C terminal His10 and Twin Strep tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5509)
  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 5509)
  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- AVITAG-CTH10STII- -GG
FEATURES             Location/Qualifiers
     source          1..5509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..65
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        46..64
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    65..89
                     /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             172..216
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             226..246
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             247..276
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(286..309,310..345,346..369)
                     /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             419..436
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(489..507)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      503..550
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      587..1042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(674..693)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     884..905
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1134..1949)
                     /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(1134..1949)
                     /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     1857..1876
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1941..1960
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2071..2659
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2560..2579
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2813..2830
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2845..2984
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2910..2929)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3070..3092
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3086..3277)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4086..5168)
                     /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     5130..5149
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5169..5246)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5452..5471
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
       61 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aaccttaaga
      121 aggagatata cacatgagag accgcggata attcgggtct cactcggatc aggcctgaac
      181 gatatttttg aagcgcagaa aattgaatgg catgaaggtt ctggagagaa cctctacttc
      241 caatcgcacc atcatcacca ccatcatcat catcacggtt ctggttggag tcacccacaa
      301 ttcgagaaag gtggtggctc tggtggcggt tcaggcggct ccgcctggtc ccatccacag
      361 tttgaaaagt gaggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca
      421 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      481 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
      541 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
      601 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
      661 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
      721 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
      781 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
      841 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
      901 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
      961 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1021 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1081 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     1141 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     1201 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1261 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     1321 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     1381 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     1441 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     1501 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     1561 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     1621 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     1681 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     1741 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     1801 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     1861 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     1921 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     1981 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2041 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2101 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2161 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2221 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2281 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2341 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2401 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2461 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2521 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2581 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2641 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2701 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2761 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2821 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     2881 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     2941 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3001 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3061 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3121 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3181 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3241 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     3301 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3361 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     3421 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     3481 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     3541 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     3601 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     3661 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     3721 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     3781 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     3841 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     3901 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     3961 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4021 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4081 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4141 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4201 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4261 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     4321 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     4381 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4441 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4501 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4561 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4621 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4681 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4741 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4801 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4861 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     4921 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     4981 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5041 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5101 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5161 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5221 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     5281 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     5341 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     5401 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     5461 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcc
//
