LOCUS Exported 3363 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Contributes the coding sequence for ECFP as the 5'-module during MultiSite Gateway-cloning of chimeric cDNAs encoding three-part fusion proteins.. ACCESSION . VERSION . KEYWORDS pDONR P4-P1R-ECFP 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3363 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 694..1407 /codon_start=1 /product="enhanced CFP" /label=ECFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL 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 caagttcagc 781 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 841 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctggggcgtg 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 catcagccac 1141 aacgtctata tcaccgccga caagcagaag aacggcatca aggccaactt 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 //