LOCUS       sequence-426299-        5519 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal OmpA SEC 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 5519)
  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 5519)
  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-OmpA-NHSTIIT-GG
FEATURES             Location/Qualifiers
     source          1..5519
                     /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)"
     sig_peptide     131..193
                     /codon_start=1
                     /product="signal peptide from the E. coli outer membrane 
                     protein OmpA"
                     /label=OmpA signal peptide
                     /translation="MKKTAIAIAVALAGFATVAQA"
     CDS             197..214
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(230..253,254..289,290..313)
                     /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             338..358
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             426..443
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(496..514)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      510..557
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      594..1049
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(681..700)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     891..912
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1141..1956)
                     /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(1141..1956)
                     /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     1864..1883
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1948..1967
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2078..2666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2567..2586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2820..2837
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2852..2991
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2917..2936)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3077..3099
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3093..3284)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4093..5175)
                     /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     5137..5156
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5176..5253)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5459..5478
                     /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 atgaagaaaa ccgcgattgc gattgcagtt gccttagcag gctttgctac
      181 ggtagcacaa gcgatgcacc atcatcatca tcattcttct ggtgcgagct ggagtcaccc
      241 acaattcgag aaaggtggtg gctctggtgg cggttcaggc ggctccgcct ggtcccatcc
      301 acagtttgaa aagggttctg gtgtagatct gggtaccgag aacctgtact tccaatccat
      361 gagagaccgc ggataattcg ggtctcactc gagcacccgt cgacaagctt gcggccgcac
      421 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
      481 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
      541 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
      601 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
      661 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
      721 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
      781 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
      841 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
      901 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
      961 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1021 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     1081 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1141 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1201 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1261 taggatggca agatcctggt atcggtctgc tattccgact cgtccaacat caatacaacc
     1321 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     1381 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     1441 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     1501 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     1561 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     1621 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     1681 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     1741 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     1801 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     1861 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     1921 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     1981 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     2041 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2101 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2161 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2221 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2281 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2341 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2401 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2461 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2521 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2581 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2641 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     2701 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2761 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2821 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     2881 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     2941 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3001 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3061 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3121 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3181 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3241 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3301 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     3361 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     3421 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     3481 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     3541 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     3601 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     3661 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     3721 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     3781 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     3841 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gtggtcctcg
     3901 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     3961 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4021 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4081 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4141 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4201 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4261 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4321 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     4381 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     4441 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     4501 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     4561 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     4621 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     4681 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     4741 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     4801 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     4861 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     4921 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     4981 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5041 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5101 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5161 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5221 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5281 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     5341 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     5401 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     5461 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacg
//
