LOCUS       sequence-426304-        6470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal His6 MBP tag and TEV 
            Cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6470)
  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 6470)
  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-HIS6-MBP-TEV-GG
FEATURES             Location/Qualifiers
     source          1..6470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     31..50
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        31..49
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    50..74
                     /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             89..111
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             128..145
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1277..1297
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1365..1382
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1435..1453)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1449..1496
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1533..1988
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1620..1639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1830..1851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2080..2895)
                     /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(2080..2895)
                     /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     2803..2822
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2887..2906
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3017..3605
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3506..3525
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3759..3776
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3791..3930
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3856..3875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4016..4038
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4032..4223)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5032..6114)
                     /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     6076..6095
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6115..6192)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6398..6417
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
       61 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat
      121 gaagatccac catcatcatc atcacgaaga gggtaagctg gtgatctgga tcaatgggga
      181 taaggggtac aacggcctgg cagaggtcgg taagaagttt gagaaggaca cagggattaa
      241 ggtgactgtt gagcaccctg ataagctgga agaaaagttc ccgcaggttg cagccaccgg
      301 agacggccct gacatcattt tctgggcaca cgaccgcttc ggtgggtacg cgcaatctgg
      361 actgttggct gaaattacgc ccgacaaggc attccaagac aaattatacc ctttcacgtg
      421 ggatgctgtc cggtataacg ggaagctgat agcgtatccg attgcggtag aggcgttgtc
      481 attaatctat aacaaggatt tattacccaa tccgcccaaa acttgggaag agatcccagc
      541 gttggacaag gagttgaagg caaagggtaa aagcgccctc atgttcaacc ttcaagaacc
      601 gtattttacg tggccattaa tcgccgctga cggtgggtac gcattcaagt acgagaacgg
      661 aaagtacgat atcaaggatg ttggggttga caacgctggg gccaaagcgg gcttaacgtt
      721 cctcgttgac ctcataaaga acaagcacat gaacgcggat acggactact cgatagctga
      781 agctgctttt aacaaaggcg agactgctat gaccattaac ggaccttggg cgtggtccaa
      841 cattgacaca tcaaaggtga actacggggt tacagtactt ccgaccttta aagggcaacc
      901 tagcaagcca ttcgtgggag tcctgtcagc cggaattaat gcggcgtcac cgaacaagga
      961 gttagcgaag gagtttcttg agaactattt acttaccgac gagggcttgg aagccgttaa
     1021 caaggacaag ccacttgggg cggtagcatt aaaatcgtac gaagaagaat tggcgaagga
     1081 cccgcggatc gccgcgacga tggaaaatgc ccagaagggc gagattatgc cgaacattcc
     1141 ccaaatgtct gcattctggt atgcagttcg cacggcagtg ataaacgcag ccagcggtcg
     1201 tcaaacggtg gacgaggctc ttaaggatgc ccaaacgaac tccataacat cgctgtacaa
     1261 gaaggctgga tctggtgaga acctgtactt ccaatccatg agagaccgcg gataattcgg
     1321 gtctcactcg agcacccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     1381 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1441 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1501 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1561 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1621 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1681 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1741 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1801 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1861 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1921 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1981 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2041 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     2101 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     2161 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     2221 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     2281 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     2341 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     2401 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     2461 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2521 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     2581 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     2641 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     2701 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     2761 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2821 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     2881 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2941 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3001 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3061 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3781 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3841 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3901 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3961 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     4021 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     4081 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     4141 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     4201 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4261 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4321 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4381 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4441 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4501 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4561 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4621 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4681 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4741 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4801 aggccgatca tcgtcgcgct ccagcgaaag tggtcctcgc cgaaaatgac ccagagcgct
     4861 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4921 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4981 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5041 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5101 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     5161 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     5221 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5281 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5341 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5401 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5461 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5521 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5581 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5641 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5701 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5761 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5821 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5881 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5941 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     6001 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6061 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     6121 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     6181 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6241 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6301 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6361 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6421 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
//
