LOCUS       sequence-445940-        2583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal GFP-TEV tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2583)
  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 2583)
  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)_GFP-TEV
FEATURES             Location/Qualifiers
     source          1..2583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             38..748
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             38..748
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(77..98)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(338..357)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     685..706
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             749..769
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      773..782
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        955..1057
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1058..1717
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1124..1143)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2112..2468
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(2521..2554)
                     /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 tcaggcgagc aagggcgagg agctgttcac
       61 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
      121 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
      181 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      241 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      301 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
      361 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
      421 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
      481 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
      541 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
      601 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
      661 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
      721 cactctcggc atggacgagc tgtacaagga aaacctgtat ttccagggag cagccgccat
      781 ggagacccac tgcttgagcc tagaagatcc ggctgctaac aaagcccgaa aggaagctga
      841 gttggctgct gtcaccgctg agcaataact atcataaccc ctaggtgcca tttcattacc
      901 tctttctccg cacccgacat agatctgggc caacttttgg cgaaaatgag acgttgatcg
      961 gcacgtaaga gggtccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt
     1021 tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     1081 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     1141 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     1201 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     1261 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     1321 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     1381 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     1441 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     1501 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     1561 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1621 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1681 acaacagtac tgcgatgagt ggcagggtgg ggcgtaattt ttttaaggca gttattggtg
     1741 cccttaaacg cctggttgct acgcctgaat aagtgataat aagcggatga atggcagaaa
     1801 ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg gtcgttaaat agccgcttat
     1861 gtctattgct ggtttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca
     1921 aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc
     1981 atttattctg cctcccagct gacattcatc cggggtcagc accgtttctg cggactggct
     2041 ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgaa
     2101 gctaattccc atgtcagccg ttaagtgttc ctgtgtcact caaaattgct ttgagaggct
     2161 ctaagggctt ctcagtgcgt tacatccctg gcttgttgtc cacaaccgtt aaaccttaaa
     2221 agctttaaaa gccttatata ttcttttttt tcttataaaa cttaaaacct tagaggctat
     2281 ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt acgtgaaaca
     2341 tgagagctta gtacgttagc catgagagct tagtacgtta gccatgaggg tttagttcgt
     2401 taaacatgag agcttagtac gttaaacatg agagcttagt acgtgaaaca tgagagctta
     2461 gtacgtacta tcaacaggtt gaactgctga tcaacagatc ctctacgcgg ccgcggtacc
     2521 ataacttcgt atagcataca ttatacgaag ttatctgcca ggcacatggg ttttactagt
     2581 atc
//
