LOCUS       sequence-426144-        6452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning of target gene with N terminal MBP tag and TEV cleavage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6452)
  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 6452)
  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-MBP-TEV-GG
FEATURES             Location/Qualifiers
     source          1..6452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        51..69
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    70..94
                     /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             109..131
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1279..1299
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1367..1384
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1437..1455)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1451..1498
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1535..1990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1622..1641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1832..1853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2082..2897)
                     /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(2082..2897)
                     /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     2805..2824
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2889..2908
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3019..3607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3508..3527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3761..3778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3793..3932
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3858..3877)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4018..4040
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4034..4225)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5034..6116)
                     /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     6078..6097
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6117..6194)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6400..6419
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
       61 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
      121 taagaaggag atatacatat gaagattgaa gagggtaagc tggtaatatg gattaacggg
      181 gacaagggct ataacgggct tgctgaggtg ggaaagaagt tcgaaaagga cacgggcatt
      241 aaggtgaccg tagagcaccc agataaactc gaggagaagt tcccacaagt tgccgcaact
      301 ggcgacggcc cggacatcat tttctgggcg cacgaccgct tcggcggata cgcccaaagt
      361 ggcctgctgg ctgagatcac gccggataag gcgtttcaag ataagcttta tccctttacc
      421 tgggacgcgg ttcgctacaa tggtaagtta atagcatatc ccattgcggt ggaagcatta
      481 tctcttatct acaataaaga cttacttcct aacccaccta agacttggga agagatccct
      541 gcactggaca aggaacttaa ggctaaagga aaatcagccc tcatgttcaa tcttcaagag
      601 ccatatttta cgtggcctct gatagccgcg gatggcggat atgcatttaa atatgagaat
      661 gggaagtatg atatcaagga cgtaggggtt gacaacgcgg gagcgaaggc ggggttgact
      721 ttccttgtgg acttaatcaa gaacaagcac atgaacgctg acactgacta ctccatagcc
      781 gaagcggctt tcaataaggg agagacggca atgacaatca atggtccgtg ggcatggagt
      841 aatatcgaca cttcaaaagt taattacggc gtcacagttt taccaacgtt taaaggtcaa
      901 cccagtaaac ccttcgtggg tgtacttagc gctgggataa atgccgccag cccaaacaag
      961 gagctggcga aggagttcct cgagaactac cttttgacgg atgaaggcct tgaggcagtc
     1021 aacaaggaca agccactggg tgctgttgca ttgaaatcat acgaagagga gctcgcgaag
     1081 gacccgcgca tcgccgcgac tatggagaat gcccaaaagg gcgagattat gccgaatata
     1141 cctcaaatgt cagccttctg gtacgcggtt cgtacggctg tcataaacgc ggcttctggc
     1201 agacagacgg tggacgaagc cttgaaagat gctcaaacca actccattac gtcactttac
     1261 aagaaggcgg gcagcggaga gaacctgtac ttccaatcca tgagagaccg cggataattc
     1321 gggtctcact cgagcacccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     1381 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1441 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1501 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1561 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1621 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1681 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1741 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1801 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1861 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1921 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1981 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2041 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     2101 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2161 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2221 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2281 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2341 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2401 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2461 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2521 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2581 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2641 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2701 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2761 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2821 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2881 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2941 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3001 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3061 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3121 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3661 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3721 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3781 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3841 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3901 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3961 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4021 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     4081 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     4141 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     4201 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4261 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4321 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4381 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4441 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4501 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4561 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4621 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4681 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4741 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4801 acaggccgat catcgtcgcg ctccagcgaa agtggtcctc gccgaaaatg acccagagcg
     4861 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4921 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4981 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     5041 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5101 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     5161 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     5221 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5281 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5341 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5401 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5461 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5521 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5581 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5641 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5701 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5761 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5821 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5881 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5941 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     6001 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     6061 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     6121 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     6181 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6241 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6301 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6361 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6421 gccagcaacc gcacctgtgg cgccggtgat gc
//
