LOCUS       sequence-445945-        2001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal His10-TwinStrep-3C tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2001)
  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 2001)
  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)_His10-TS-3C
FEATURES             Location/Qualifiers
     source          1..2001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        29..131
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             132..791
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(198..217)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      1186..1542
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(1595..1628)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1695..1724
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1740..1763
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1800..1823
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1824..1847
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
ORIGIN
        1 gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagagggtcc aactttcacc
       61 ataatgaaat aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta
      121 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
      181 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
      241 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
      301 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa
      361 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
      421 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
      481 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
      541 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
      601 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
      661 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
      721 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
      781 gtggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt tgctacgcct
      841 gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg acccggtcgt
      901 cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta ccggtttatt
      961 gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt ttgctcaggc
     1021 tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc agctgacatt
     1081 catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca
     1141 gcccttgcgc cctgagtgct tgcggcagcg tgaagctaat tcccatgtca gccgttaagt
     1201 gttcctgtgt cactcaaaat tgctttgaga ggctctaagg gcttctcagt gcgttacatc
     1261 cctggcttgt tgtccacaac cgttaaacct taaaagcttt aaaagcctta tatattcttt
     1321 tttttcttat aaaacttaaa accttagagg ctatttaagt tgctgattta tattaatttt
     1381 attgttcaaa catgagagct tagtacgtga aacatgagag cttagtacgt tagccatgag
     1441 agcttagtac gttagccatg agggtttagt tcgttaaaca tgagagctta gtacgttaaa
     1501 catgagagct tagtacgtga aacatgagag cttagtacgt actatcaaca ggttgaactg
     1561 ctgatcaaca gatcctctac gcggccgcgg taccataact tcgtatagca tacattatac
     1621 gaagttatct gccaggcaca tgggttttac tagtatcgat tcgcgaccta ctccggaata
     1681 ttaggtctca ggcgcaccat caccatcacc atcaccatca ccatggtgga tctagcgctt
     1741 ggagccaccc gcagttcgag aaaggtggag gttccggagg tggatcggga ggtggatcgt
     1801 ggagccaccc gcagttcgaa aaactggaag ttctgttcca ggggcccgca gccatggaga
     1861 cccactgctt gagcctagaa gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1921 ctgctgtcac cgctgagcaa taactatcat aacccctagg tgccatttca ttacctcttt
     1981 ctccgcaccc gacatagatc t
//
