LOCUS       sequence-445942-        2556 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  4G-cloning donor for N-terminal GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2556)
  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 2556)
  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
FEATURES             Location/Qualifiers
     source          1..2556
                     /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"
     promoter        928..1030
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1031..1690
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1097..1116)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2085..2441
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    complement(2494..2527)
                     /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 tgtacaaggc catggagacc cactgcttga gcctagaaga
      781 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgtcaccg ctgagcaata
      841 actatcataa cccctaggtg ccatttcatt acctctttct ccgcacccga catagatctg
      901 ggccaacttt tggcgaaaat gagacgttga tcggcacgta agagggtcca actttcacca
      961 taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa
     1021 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     1081 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     1141 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     1201 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat
     1261 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga
     1321 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct
     1381 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg
     1441 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga
     1501 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta
     1561 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga
     1621 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg
     1681 tggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctggtt gctacgcctg
     1741 aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga cccggtcgtc
     1801 ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac cggtttattg
     1861 actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt tgctcaggct
     1921 ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca gctgacattc
     1981 atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag
     2041 cccttgcgcc ctgagtgctt gcggcagcgt gaagctaatt cccatgtcag ccgttaagtg
     2101 ttcctgtgtc actcaaaatt gctttgagag gctctaaggg cttctcagtg cgttacatcc
     2161 ctggcttgtt gtccacaacc gttaaacctt aaaagcttta aaagccttat atattctttt
     2221 ttttcttata aaacttaaaa ccttagaggc tatttaagtt gctgatttat attaatttta
     2281 ttgttcaaac atgagagctt agtacgtgaa acatgagagc ttagtacgtt agccatgaga
     2341 gcttagtacg ttagccatga gggtttagtt cgttaaacat gagagcttag tacgttaaac
     2401 atgagagctt agtacgtgaa acatgagagc ttagtacgta ctatcaacag gttgaactgc
     2461 tgatcaacag atcctctacg cggccgcggt accataactt cgtatagcat acattatacg
     2521 aagttatctg ccaggcacat gggttttact agtatc
//
