LOCUS       sequence-70631-e        4058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contributes an IRES-ECFP cassette as the 3'-module during MultiSite 
            Gateway cloning of a bi-cistronic mRNA shared with a two-part fusion
            protein encoded by the 5′- and middle modules..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4058)
  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 4058)
  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 P2R-P3-IRES_ECFP
FEATURES             Location/Qualifiers
     source          1..4058
                     /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    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    join(786..1360,1361..1363,1364..1372)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(953..970)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1180..1199
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(1373..1375,1376..1378,1379..2089)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1418..1439)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1679..1698)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2026..2047
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2108..2134
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2138..2155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    complement(2167..2262)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2299..2318)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2300..2318)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2323..2339)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2323..2339)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2441..2460)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2452..3261
                     /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(2519..2538)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3408..3996
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3897..3916
                     /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 tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtggaca ttctgcagtc gacggtaccg cgggcccggg
      781 atccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc
      841 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
      901 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
      961 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     1021 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     1081 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     1141 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     1201 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     1261 cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     1321 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatggtgag
     1381 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1441 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1501 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1561 caccctgacc tggggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1621 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1681 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1741 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1801 gtacaactac atcagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa
     1861 ggccaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1921 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1981 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2041 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ctagagtcga
     2101 ccagctgtat ccatacgacg tgccagatta cgctggacac catcaccacc atcacgcctg
     2161 actgaacaac tttattatac aaagttggca ttataaaaaa gcattgctta tcaatttgtt
     2221 gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttggagctcc atggtagcgt
     2281 taacgcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg
     2341 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
     2401 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     2461 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     2521 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     2581 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     2641 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     2701 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
     2761 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     2821 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     2881 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     2941 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     3001 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     3061 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     3121 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     3181 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     3241 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     3301 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg
     3361 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3421 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3481 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3541 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3601 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3661 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3721 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3781 agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3841 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3901 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3961 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4021 cggttcctgg ccttttgctg gccttttgct cacatgtt
//
