LOCUS       sequence-445955-        1956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal His6-FLAG3-TEV tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 1956)
  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 1956)
  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-FLAG3-TEV
FEATURES             Location/Qualifiers
     source          1..1956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      192..548
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(601..634)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             701..718
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             719..784
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             785..805
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      809..818
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        991..1093
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1094..1753
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1160..1179)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc
       61 atttattctg cctcccagct gacattcatc cggggtcagc accgtttctg cggactggct
      121 ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgaa
      181 gctaattccc atgtcagccg ttaagtgttc ctgtgtcact caaaattgct ttgagaggct
      241 ctaagggctt ctcagtgcgt tacatccctg gcttgttgtc cacaaccgtt aaaccttaaa
      301 agctttaaaa gccttatata ttcttttttt tcttataaaa cttaaaacct tagaggctat
      361 ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt acgtgaaaca
      421 tgagagctta gtacgttagc catgagagct tagtacgtta gccatgaggg tttagttcgt
      481 taaacatgag agcttagtac gttaaacatg agagcttagt acgtgaaaca tgagagctta
      541 gtacgtacta tcaacaggtt gaactgctga tcaacagatc ctctacgcgg ccgcggtacc
      601 ataacttcgt atagcataca ttatacgaag ttatctgcca ggcacatggg ttttactagt
      661 atcgattcgc gacctactcc ggaatattag gtctcaggcg caccatcacc atcaccatga
      721 ctacaaagac catgacggtg attataaaga tcatgatatc gattacaagg atgacgatga
      781 caaggaaaac ctgtatttcc agggagcagc cgccatggag acccactgct tgagcctaga
      841 agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgtca ccgctgagca
      901 ataactatca taacccctag gtgccatttc attacctctt tctccgcacc cgacatagat
      961 ctgggccaac ttttggcgaa aatgagacgt tgatcggcac gtaagagggt ccaactttca
     1021 ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc
     1081 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     1141 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     1201 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     1261 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc
     1321 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     1381 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     1441 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     1501 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     1561 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata
     1621 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg
     1681 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     1741 gggtggggcg taattttttt aaggcagtta ttggtgccct taaacgcctg gttgctacgc
     1801 ctgaataagt gataataagc ggatgaatgg cagaaattcg aaagcaaatt cgacccggtc
     1861 gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct attgctggtt taccggttta
     1921 ttgactaccg gaagcagtgt gaccgtgtgc ttctca
//
