LOCUS       sequence-445970-        1999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for C-terminal 3C-TwinSTrep-His10 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 1999)
  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 1999)
  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(Ct)_3C-TS-His10
FEATURES             Location/Qualifiers
     source          1..1999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             40..63
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             79..102
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             139..162
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             163..192
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        371..473
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             474..1133
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(540..559)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      1528..1884
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(1937..1970)
                     /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 tcaggatccc tggaagttct gttccagggg
       61 cccggtggat ctagcgcttg gagccacccg cagttcgaga aaggtggagg ttccggaggt
      121 ggatcgggag gtggatcgtg gagccacccg cagttcgaaa aacaccatca ccatcaccat
      181 caccatcacc attaagggag acccactgct tgagcctaga agatccggct gctaacaaag
      241 cccgaaagga agctgagttg gctgctgtca ccgctgagca ataactatca taacccctag
      301 gtgccatttc attacctctt tctccgcacc cgacatagat ctgggccaac ttttggcgaa
      361 aatgagacgt tgatcggcac gtaagagggt ccaactttca ccataatgaa ataagatcac
      421 taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
      481 aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg
      541 aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg
      601 cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc
      661 ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg
      721 tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
      781 catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag
      841 atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt
      901 ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata
      961 tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg
     1021 tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
     1081 gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggtggggcg taattttttt
     1141 aaggcagtta ttggtgccct taaacgcctg gttgctacgc ctgaataagt gataataagc
     1201 ggatgaatgg cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg
     1261 ttaaatagcc gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt
     1321 gaccgtgtgc ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat
     1381 aattgacgat atgatcattt attctgcctc ccagctgaca ttcatccggg gtcagcaccg
     1441 tttctgcgga ctggctttct acgtgttccg cttcctttag cagcccttgc gccctgagtg
     1501 cttgcggcag cgtgaagcta attcccatgt cagccgttaa gtgttcctgt gtcactcaaa
     1561 attgctttga gaggctctaa gggcttctca gtgcgttaca tccctggctt gttgtccaca
     1621 accgttaaac cttaaaagct ttaaaagcct tatatattct tttttttctt ataaaactta
     1681 aaaccttaga ggctatttaa gttgctgatt tatattaatt ttattgttca aacatgagag
     1741 cttagtacgt gaaacatgag agcttagtac gttagccatg agagcttagt acgttagcca
     1801 tgagggttta gttcgttaaa catgagagct tagtacgtta aacatgagag cttagtacgt
     1861 gaaacatgag agcttagtac gtactatcaa caggttgaac tgctgatcaa cagatcctct
     1921 acgcggccgc ggtaccataa cttcgtatag catacattat acgaagttat ctgccaggca
     1981 catgggtttt actagtatc
//
