LOCUS Exported 4268 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION TOPO construct expressing mKate2 for generation of GMAP-compatible gene. ACCESSION . VERSION . KEYWORDS TOPO mKate2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4268) AUTHORS Akama-Garren EH, Joshi NS, Tammela T, Chang GP, Wagner BL, Lee DY, Rideout Iii WM, Papagiannakopoulos T, Xue W, Jacks T TITLE A Modular Assembly Platform for Rapid Generation of DNA Constructs. JOURNAL Sci Rep. 2016 Feb 18;6:16836. doi: 10.1038/srep16836. PUBMED 26887506 REFERENCE 2 (bases 1 to 4268) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4268 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 239..256 /label=SP6 /note="SP6 promoter, forward primer" CDS 332..1030 /codon_start=1 /product="monomeric far-red fluorescent protein (Shcherbo et al., 2009)" /label=mKate2 /note="mammalian codon-optimized" /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY CDLPSKLGHR" primer_bind complement(1155..1174) /label=T7 /note="T7 promoter, forward primer" promoter complement(1156..1174) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1181..1198) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1181..1197) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1190..1212) /label=M13/pUC Forward /note="In lacZ gene" CDS 1335..1637 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 1516..1535 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1986..2780 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(2040..2059) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2650..2669 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2981..2990 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2987..3361 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 3499..4087 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3988..4007 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4241..4258 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcct 301 aactcgaacg ctagctgtgc gatcgtttac catggtgagc gagctgatta aggagaacat 361 gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga 421 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggcgg tcgagggcgg 481 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcaaaacctt 541 catcaaccac acccagggca tccccgactt ctttaagcag tccttccccg agggcttcac 601 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag 661 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa 721 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc tccaccgaga ccctgtaccc 781 cgctgacggc ggcctggaag gcagagccga catggccctg aagctcgtgg gcgggggcca 841 cctgatctgc aacttgaaga ccacatacag atccaagaaa cccgctaaga acctcaagat 901 gcccggcgtc tactatgtgg acagaagact ggaaagaatc aaggaggccg acaaagagac 961 ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg 1021 gcacagatga aaacgttgtt gtttggggtt gaattactct ggatccacta gtaacggccg 1081 ccagtgtgct ggaattctgc agatatccat cacactggcg gccgctcgag catgcatcta 1141 gagggcccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac 1201 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 1261 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 1321 gcctatacgt acggcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt 1381 ttgtggatgt acagagtgat attattgaca cgccggggcg acggatggtg atccccctgg 1441 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg 1501 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg 1561 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga 1621 tgttctgggg aatataaatg tcaggcatga gattatcaaa aaggatcttc acctagatcc 1681 ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact 1741 gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc 1801 ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag 1861 ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttctcgc 1921 cgccaaggat ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt 1981 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 2041 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 2101 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 2161 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 2221 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 2281 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 2341 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 2401 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 2461 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc 2521 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 2581 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 2641 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 2701 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 2761 ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg tattttctcc 2821 ttacgcatct gtgcggtatt tcacaccgca tacaggtggc acttttcggg gaaatgtgcg 2881 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 2941 ataaccctga taaatgcttc aataatagca cgtgaggagg gccaccatgg ccaagttgac 3001 cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga 3061 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga 3121 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg 3181 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa 3241 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga 3301 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg 3361 acacgtgcta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 3421 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 3481 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 3541 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 3601 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 3661 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 3721 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 3781 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 3841 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 3901 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 3961 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 4021 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 4081 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 4141 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 4201 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 4261 agcggaag //