LOCUS       sequence-445956-        1932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal His6-FLAG3 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 1932)
  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 1932)
  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
FEATURES             Location/Qualifiers
     source          1..1932
                     /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"
     promoter        967..1069
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1070..1729
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1136..1155)
                     /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 caaggaagcc atggagaccc actgcttgag cctagaagat ccggctgcta acaaagcccg
      841 aaaggaagct gagttggctg ctgtcaccgc tgagcaataa ctatcataac ccctaggtgc
      901 catttcatta cctctttctc cgcacccgac atagatctgg gccaactttt ggcgaaaatg
      961 agacgttgat cggcacgtaa gagggtccaa ctttcaccat aatgaaataa gatcactacc
     1021 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     1081 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     1141 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     1201 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     1261 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     1321 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     1381 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     1441 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1501 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1561 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1621 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     1681 gcttaatgaa ttacaacagt actgcgatga gtggcagggt ggggcgtaat ttttttaagg
     1741 cagttattgg tgcccttaaa cgcctggttg ctacgcctga ataagtgata ataagcggat
     1801 gaatggcaga aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa
     1861 atagccgctt atgtctattg ctggtttacc ggtttattga ctaccggaag cagtgtgacc
     1921 gtgtgcttct ca
//
