LOCUS       sequence-70559-e        3363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contributes the coding sequence for EGFP as the 5'-module during 
            MultiSite Gateway-cloning of chimeric cDNAs encoding three-part 
            fusion proteins..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3363)
  AUTHORS   Buj R, Iglesias N, Planas AM, Santalucia T
  TITLE     A plasmid toolkit for cloning chimeric cDNAs encoding customized 
            fusion proteins into any Gateway destination expression vector.
  JOURNAL   BMC Mol Biol. 2013 Aug 20;14(1):18. doi: 10.1186/1471-2199-14-18.
  PUBMED    23957834
REFERENCE   2  (bases 1 to 3363)
  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: "BMC Mol 
            Biol."; date: "2013-08-20"; volume: "14(1):18. doi"; pages: " 
            10.1186/1471-2199-14-18"
COMMENT     Sequence Label: pDONR P4-P1R-EGFP
FEATURES             Location/Qualifiers
     source          1..3363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    593..688
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(694..696,697..699,700..1407)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKVSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(739..760)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1000..1019)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1347..1368
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1408..1532
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1604..1623)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1605..1623)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1628..1644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1628..1644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1746..1765)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1757..2566
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1824..1843)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2713..3301
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3202..3221
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccgc gttaacgcta ccatggagct ccaaataatg
      601 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgctttttt
      661 ataatgccaa ctttgtatag aaaagttgcc accatggtga gcaagggcga ggagctgttc
      721 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caaggtcagc
      781 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      841 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      901 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      961 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1021 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1081 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1141 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1201 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1261 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1321 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1381 atcactctcg gcatggacga gctgtacaca agtttgtaca aaaaagttga acgagaaacg
     1441 taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa
     1501 tactgtaaaa cacaacatat gcagtcacta tgaaccaact acttagatgg tattagtgac
     1561 ctgtagaatt cgagctctag agctgcaggg cggccgcgat atcccctata gtgagtcgta
     1621 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     1681 cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag
     1741 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     1801 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     1861 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     1921 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     1981 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     2041 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     2101 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     2161 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     2221 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     2281 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     2341 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     2401 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     2461 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     2521 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     2581 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     2641 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     2701 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2761 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2821 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2881 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2941 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3001 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3061 ggagcgaacg acctacaccg aactgagata cctacagcgt gagcattgag aaagcgccac
     3121 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3181 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3241 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3301 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3361 gtt
//
