LOCUS       sequence-445935-        2581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for C-terminal 3C-GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2581)
  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 2581)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..2581
                     /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             64..777
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             64..774
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(103..124)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(364..383)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     711..732
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        953..1055
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1056..1715
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1122..1141)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2110..2466
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(2519..2552)
                     /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 cccagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      121 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
      181 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
      241 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
      301 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
      361 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
      421 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
      481 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
      541 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
      601 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
      661 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
      721 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaggg
      781 agacccactg cttgagccta gaagatccgg ctgctaacaa agcccgaaag gaagctgagt
      841 tggctgctgt caccgctgag caataactat cataacccct aggtgccatt tcattacctc
      901 tttctccgca cccgacatag atctgggcca acttttggcg aaaatgagac gttgatcggc
      961 acgtaagagg gtccaacttt caccataatg aaataagatc actaccgggc gtattttttg
     1021 agttatcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     1081 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     1141 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     1201 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     1261 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     1321 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     1381 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     1441 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     1501 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     1561 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     1621 ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     1681 aacagtactg cgatgagtgg cagggtgggg cgtaattttt ttaaggcagt tattggtgcc
     1741 cttaaacgcc tggttgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt
     1801 cgaaagcaaa ttcgacccgg tcgtcggttc agggcagggt cgttaaatag ccgcttatgt
     1861 ctattgctgg tttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa
     1921 tgcctgaggc cagtttgctc aggctctccc cgtggaggta ataattgacg atatgatcat
     1981 ttattctgcc tcccagctga cattcatccg gggtcagcac cgtttctgcg gactggcttt
     2041 ctacgtgttc cgcttccttt agcagccctt gcgccctgag tgcttgcggc agcgtgaagc
     2101 taattcccat gtcagccgtt aagtgttcct gtgtcactca aaattgcttt gagaggctct
     2161 aagggcttct cagtgcgtta catccctggc ttgttgtcca caaccgttaa accttaaaag
     2221 ctttaaaagc cttatatatt cttttttttc ttataaaact taaaacctta gaggctattt
     2281 aagttgctga tttatattaa ttttattgtt caaacatgag agcttagtac gtgaaacatg
     2341 agagcttagt acgttagcca tgagagctta gtacgttagc catgagggtt tagttcgtta
     2401 aacatgagag cttagtacgt taaacatgag agcttagtac gtgaaacatg agagcttagt
     2461 acgtactatc aacaggttga actgctgatc aacagatcct ctacgcggcc gcggtaccat
     2521 aacttcgtat agcatacatt atacgaagtt atctgccagg cacatgggtt ttactagtat
     2581 c
//
