LOCUS       sequence-445966-        2562 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal His6-GST-TEV tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2562)
  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 2562)
  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-GST-TEV
FEATURES             Location/Qualifiers
     source          1..2562
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             38..55
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             59..709
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEVHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     667..689
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             728..748
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      752..761
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        934..1036
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1037..1696
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1103..1122)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2091..2447
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(2500..2533)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gattcgcgac ctactccgga atattaggtc tcaggcgcac catcaccatc accatggatc
       61 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      121 atatcttgaa gaaaaatatg aagtgcattt gtatgagcgc gatgaaggtg ataaatggcg
      181 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      241 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      301 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      361 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      421 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      481 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      541 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      601 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
      661 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatggttc
      721 aactagtgaa aacctgtatt tccagggagc agccgccatg gagacccact gcttgagcct
      781 agaagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg tcaccgctga
      841 gcaataacta tcataacccc taggtgccat ttcattacct ctttctccgc acccgacata
      901 gatctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag ggtccaactt
      961 tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg
     1021 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     1081 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     1141 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     1201 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat
     1261 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     1321 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     1381 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     1441 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     1501 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     1561 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     1621 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     1681 gcagggtggg gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggttgcta
     1741 cgcctgaata agtgataata agcggatgaa tggcagaaat tcgaaagcaa attcgacccg
     1801 gtcgtcggtt cagggcaggg tcgttaaata gccgcttatg tctattgctg gtttaccggt
     1861 ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct
     1921 caggctctcc ccgtggaggt aataattgac gatatgatca tttattctgc ctcccagctg
     1981 acattcatcc ggggtcagca ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt
     2041 tagcagccct tgcgccctga gtgcttgcgg cagcgtgaag ctaattccca tgtcagccgt
     2101 taagtgttcc tgtgtcactc aaaattgctt tgagaggctc taagggcttc tcagtgcgtt
     2161 acatccctgg cttgttgtcc acaaccgtta aaccttaaaa gctttaaaag ccttatatat
     2221 tctttttttt cttataaaac ttaaaacctt agaggctatt taagttgctg atttatatta
     2281 attttattgt tcaaacatga gagcttagta cgtgaaacat gagagcttag tacgttagcc
     2341 atgagagctt agtacgttag ccatgagggt ttagttcgtt aaacatgaga gcttagtacg
     2401 ttaaacatga gagcttagta cgtgaaacat gagagcttag tacgtactat caacaggttg
     2461 aactgctgat caacagatcc tctacgcggc cgcggtacca taacttcgta tagcatacat
     2521 tatacgaagt tatctgccag gcacatgggt tttactagta tc
//
