LOCUS Exported 3350 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Multisite gateway vector for 3' tagging with mCherry. ACCESSION . VERSION . KEYWORDS p3E-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3350) AUTHORS Ariotti N, Rae J, Giles N, Martel N, Sierecki E, Gambin Y, Hall TE, Parton RG TITLE Ultrastructural localisation of protein interactions using conditionally stable nanobodies. JOURNAL PLoS Biol. 2018 Apr 5;16(4):e2005473. doi: 10.1371/journal.pbio.2005473. eCollection 2018 Apr. PUBMED 29621251 REFERENCE 2 (bases 1 to 3350) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3350 /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 complement(624..748) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 750..1457 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(892..910) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1160..1179 /label=mCherry-F /note="mCherry, forward primer" primer_bind 1354..1377 /label=DsRed1-C /note="DsRed1, forward primer" protein_bind complement(1459..1554) /gene="mutant version of attL" /label=attL3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(1591..1610) /label=T7 /note="T7 promoter, forward primer" promoter complement(1592..1610) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1615..1631) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1615..1631) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(1733..1752) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 1744..2553 /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(1811..1830) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 2700..3288 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3189..3208 /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 attcatagtg actgcatatg ttgtgtttta cagtattatg 661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 721 ctcgttcaac tttcttgtac aaagtggctg tgagcaaggg cgaggaggac aacatggcca 781 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt 841 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga 901 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt 961 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct 1021 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg 1081 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca 1141 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct 1201 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc 1261 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacaaggcc aagaagcccg 1321 tgcagctgcc cggcgcctac aacgtcaaca tcaagctgga catcacctcc cacaacgagg 1381 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggcggcatgg 1441 acgagctgta caagtaacca actttattat acaaagttgg cattataaaa aagcattgct 1501 tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttggagct 1561 ccatggtagc gttaacgcgg ccgcgatatc ccctatagtg agtcgtatta catggtcata 1621 gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat 1681 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt 1741 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct 1801 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 1861 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 1921 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 1981 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 2041 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 2101 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 2161 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt 2221 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 2281 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 2341 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga 2401 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct 2461 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg 2521 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac 2581 tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac 2641 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 2701 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 2761 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 2821 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 2881 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 2941 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3001 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3061 tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 3121 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 3181 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 3241 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 3301 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt //