LOCUS       sequence-426132-        5537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal SufI TatA pathway signal 
            peptide His6 Twin Strep tag and TEV cleavage .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5537)
  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 5537)
  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-SufI-NHSTIIT-GG
FEATURES             Location/Qualifiers
     source          1..5537
                     /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)."
     RBS             107..129
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             221..238
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(254..277,278..313,314..337)
                     /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..382
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             450..467
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(520..538)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      534..581
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      618..1073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(705..724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     915..936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1165..1980)
                     /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(1165..1980)
                     /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     1888..1907
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1972..1991
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2102..2690
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2591..2610
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2844..2861
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2876..3015
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2941..2960)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3101..3123
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3117..3308)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4117..5199)
                     /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     5161..5180
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5200..5277)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5483..5502
                     /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 tttaacttta
      121 agaaggagat atacatatga gcctgagtcg tcgccagttt atccaagcgt ccggcattgc
      181 cttatgcgct ggtgcagtgc cgctgaaagc ctcggcgatg caccatcatc atcatcattc
      241 ttctggtgcg agctggagtc acccacaatt cgagaaaggt ggtggctctg gtggcggttc
      301 aggcggctcc gcctggtccc atccacagtt tgaaaagggt tctggtgtag atctgggtac
      361 cgagaacctg tacttccaat ccatgagaga ccgcggataa ttcgggtctc actcgagcac
      421 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
      481 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
      541 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
      601 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
      661 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
      721 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
      781 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
      841 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
      901 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
      961 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1021 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1081 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1141 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1201 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     1261 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgctattcc
     1321 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     1381 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     1441 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     1501 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     1561 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     1621 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     1681 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     1741 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     1801 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     1861 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     1921 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     1981 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     2041 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2101 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2161 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2221 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     2281 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2341 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2401 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2461 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2521 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2581 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2641 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2701 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2761 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2821 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     2881 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     2941 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3001 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3061 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3121 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3181 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3241 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3301 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     3361 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     3421 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     3481 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     3541 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     3601 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     3661 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     3721 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     3781 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     3841 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     3901 gcgctccagc gaaagtggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     3961 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4021 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4081 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4141 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4201 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4261 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4321 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4381 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4441 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4501 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4561 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4621 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4681 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4741 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4801 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4861 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4921 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4981 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5041 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5101 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5161 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5221 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5281 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5341 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5401 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5461 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5521 tggcgccggt gatgccg
//
