LOCUS       sequence-445964-        3021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal His6-MBP-TEV tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3021)
  AUTHORS   Taschner M, Dickinson JB, Roisne-Hamelin F, Gruber S
  TITLE     4G cloning: rapid gene assembly for expression of multisubunit 
            protein complexes in diverse hosts
  JOURNAL   bioRxiv 2024.06.17.599261
REFERENCE   2  (bases 1 to 3021)
  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 
            2024.06.17.599261"
COMMENT     Sequence Label: pD(Nt)_His6-MBP-TEV
FEATURES             Location/Qualifiers
     source          1..3021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        175..277
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             278..937
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(344..363)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      1332..1688
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(1741..1774)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1841..1858
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1862..2959
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGHAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     2933..2956
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2990..3010
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      join(3014..3021,1..2)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ggagacccac tgcttgagcc tagaagatcc ggctgctaac aaagcccgaa aggaagctga
       61 gttggctgct gtcaccgctg agcaataact atcataaccc ctaggtgcca tttcattacc
      121 tctttctccg cacccgacat agatctgggc caacttttgg cgaaaatgag acgttgatcg
      181 gcacgtaaga gggtccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt
      241 tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
      301 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
      361 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
      421 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
      481 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
      541 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
      601 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
      661 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
      721 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
      781 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
      841 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
      901 acaacagtac tgcgatgagt ggcagggtgg ggcgtaattt ttttaaggca gttattggtg
      961 cccttaaacg cctggttgct acgcctgaat aagtgataat aagcggatga atggcagaaa
     1021 ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg gtcgttaaat agccgcttat
     1081 gtctattgct ggtttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca
     1141 aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc
     1201 atttattctg cctcccagct gacattcatc cggggtcagc accgtttctg cggactggct
     1261 ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgaa
     1321 gctaattccc atgtcagccg ttaagtgttc ctgtgtcact caaaattgct ttgagaggct
     1381 ctaagggctt ctcagtgcgt tacatccctg gcttgttgtc cacaaccgtt aaaccttaaa
     1441 agctttaaaa gccttatata ttcttttttt tcttataaaa cttaaaacct tagaggctat
     1501 ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt acgtgaaaca
     1561 tgagagctta gtacgttagc catgagagct tagtacgtta gccatgaggg tttagttcgt
     1621 taaacatgag agcttagtac gttaaacatg agagcttagt acgtgaaaca tgagagctta
     1681 gtacgtacta tcaacaggtt gaactgctga tcaacagatc ctctacgcgg ccgcggtacc
     1741 ataacttcgt atagcataca ttatacgaag ttatctgcca ggcacatggg ttttactagt
     1801 atcgattcgc gacctactcc ggaatattag gtctcaggcg caccatcacc atcaccatgg
     1861 aaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtca
     1921 cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc
     1981 ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat
     2041 cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac
     2101 cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa
     2161 cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga
     2221 tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa
     2281 agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct
     2341 gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga
     2401 cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa
     2461 aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg
     2521 cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt
     2581 gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg
     2641 cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct
     2701 cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg
     2761 tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac
     2821 tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg
     2881 gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc
     2941 cctgaaagac gcgcagacta attcgagctc gaacaacaac aacactagtg aaaacctgta
     3001 tttccaggga gcagccgcca t
//
