LOCUS Exported 7544 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Express pHluorin-mKate2-hLC3∆G (PK-hLC3∆G), a negative control for pHluorin-mKate2-hLC3 (PK-hLC3), in mammalian cells for monitoring autophagy. ACCESSION . VERSION . KEYWORDS pEX-PK-hLC3∆G SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7544) AUTHORS Tanida I, Ueno T, Uchiyama Y TITLE A Super-Ecliptic, pHluorin-mKate2, Tandem Fluorescent Protein-Tagged Human LC3 for the Monitoring of Mammalian Autophagy. JOURNAL PLoS One. 2014 Oct 23;9(10):e110600. doi: 10.1371/journal.pone.0110600. eCollection 2014. PUBMED 25340751 REFERENCE 2 (bases 1 to 7544) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7544 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 4..383 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" intron 654..1670 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1594..1616 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1678..1697 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 1754..2467 /codon_start=1 /product="pH-sensitive mutant of green fluorescent protein (Ng et al., 2002)" /label=superecliptic pHluorin /note="excited strongly by blue light at neutral pH but minimally fluorescent at acidic pH" /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" primer_bind complement(1784..1812) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 2407..2427 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" CDS 2486..3181 /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" CDS 3209..3580 /codon_start=1 /gene="MAP1LC3B" /product="human LC3 protein" /label=LC3B /note="Atg8 family member found on autophagosomes" /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD EDGFLYMVYASQETFMKLSV" primer_bind complement(3683..3702) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 3748..3803 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(3802..3821) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind complement(4191..4210) /label=RVprimer3 /note="pGL3 vector, forward primer" polyA_signal 4264..4312 /note="synthetic polyadenylation signal" CDS 4417..5277 /codon_start=1 /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 5477..6065 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5966..5985 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6219..6236 /label=L4440 /note="L4440 vector, forward primer" polyA_signal 6392..6440 /note="synthetic polyadenylation signal" CDS complement(6465..7064) /codon_start=1 /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" promoter complement(7095..7452) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 7109..7244 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(7163..7182) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind 7409..7429 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" ORIGIN 1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 481 cgatgggggc gggggggggg gcgccaggcg gggcggggcg gggcgagggg cggggcgggg 541 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta 601 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg 661 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc 721 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct 781 gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg 841 aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg 901 tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc 961 gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg 1021 ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 1081 gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc 1141 cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg 1201 ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc 1261 ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc 1321 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac 1381 ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta 1441 gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc 1501 gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg 1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcttaagg gtaccgatat 1741 cgctagcgcc accatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt 1801 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 1861 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 1921 atggccaaca cttgtcacta cgttaactta tggtgttcaa tgcttttcaa gatacccaga 1981 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttacg tacaggaaag 2041 aactatattt ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg 2101 tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat 2161 tcttggacac aaattggaat acaactataa cgatcaccag gtgtacatca tggcagacaa 2221 acaaaagaat ggaatcaaag ctaacttcaa aattagacac aacattgaag atggaggcgt 2281 tcaactagca gaccattatc aacaaaatac tccaattggc gatgggcccg tccttttacc 2341 agacaaccat tacctgttta caacttctac tctttcgaaa gatcccaacg aaaagagaga 2401 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 2461 atacaaaggt ggaggaggta gatccatggt gagcgagctg attaaggaga acatgcacat 2521 gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga 2581 aggcaagccc tacgagggca cccagaccat gagaatcaag gcggtcgagg gcggccctct 2641 ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcaaaa ccttcatcaa 2701 ccacacccag ggcatccccg acttctttaa gcagtccttc cccgagggct tcacatggga 2761 gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca 2821 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcccat ccaacggccc 2881 tgtgatgcag aagaaaacac tcggctggga ggcctccacc gagaccctgt accccgctga 2941 cggcggcctg gaaggcagag ccgacatggc cctgaagctc gtgggcgggg gccacctgat 3001 ctgcaacttg aagaccacat acagatccaa gaaacccgct aagaacctca agatgcccgg 3061 cgtctactat gtggacagaa gactggaaag aatcaaggag gccgacaaag agacctacgt 3121 cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacag 3181 aggtggagga ggttccggac tcagatctat gccgtcggag aagaccttca agcagcgccg 3241 caccttcgaa caaagagtag aagatgtccg acttattcga gagcagcatc caaccaaaat 3301 cccggtgata atagaacgat acaagggtga gaagcagctt cctgttctgg ataaaacaaa 3361 gttccttgta cctgaccatg tcaacatgag tgagctcatc aagataatta gaaggcgctt 3421 acagctcaat gctaatcagg ccttcttcct gttggtgaac ggacacagca tggtcagcgt 3481 ctccacacca atctcagagg tgtatgagag tgagaaagat gaagatggat tcctgtacat 3541 ggtctacgcc tcccaggaga cgttcatgaa attgtcagtg taagtcgagc ccgggagctc 3601 aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc 3661 tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga 3721 agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt 3781 gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac 3841 atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg gctgccatga 3901 acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct gtccattcct 3961 tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt tttgtgttat 4021 ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga tttttcctcc 4081 tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc ctcgacctgc 4141 agcccaagct tggccgccga ggccatgttc tggcacctgc acttgcactg gggacagcct 4201 attttgctag tttgttttgt ttcgttttgt tttgatggag agcgtatgtt agtactatcg 4261 attcacacaa aaaaccaaca cacagatgta atgaaaataa agatatttta ttgcggcctg 4321 tccaatacct cccgtacctt aatattactt acttatcctt gagagacgta ctagtaaccc 4381 tgataaatgc ttcaataata ttgaaaaagg acgagtatga gcatccaaca ttttcgtgtc 4441 gcactcattc ccttctttgc ggcattttgc ttgcctgttt ttgcacaccc cgaaacgctg 4501 gtgaaagtaa aagatgctga agatcaactg ggtgcaagag tgggctatat cgaactggat 4561 ctcaatagcg gcaagatcct tgagtctttc cgccccgaag aacgattccc gatgatgagc 4621 actttcaaag tactgctatg tggcgcggtg ttgtcccgta tagacgccgg gcaagagcag 4681 cttggtcgcc gtatacacta ctcacaaaac gacttggttg agtactcgcc ggtcacggaa 4741 aagcatctta cggatggcat gacggtaaga gaattgtgta gtgctgccat taccatgagc 4801 gacaacaccg cggccaactt acttctgaca acgatcggag gccctaagga gctgactgca 4861 tttcttcata atatgggtga tcatgtgacc cggcttgacc gctgggaacc agagttgaac 4921 gaagccatac cgaacgacga gcgtgatacc acgatgccag tagcaatggc cacaactctt 4981 cggaaactac tcactggcga acttcttact ctagcatcac gacagcagct catagactgg 5041 atggaggcgg acaaagtagc aggaccactt cttcgctcgg ccctccctgc tggctggttc 5101 attgctgaca aatcgggggc cggtgaacgc ggctctcgcg gcatcattgc tgcgctgggg 5161 cctgatggta agccctcacg aatcgtagtg atctacacga cggggagtca ggccactatg 5221 gacgaacgaa ataggcagat cgctgagatc ggtgcctcac tgatcaagca ctggtaacca 5281 ctgcagtggt ttagcatttg cggccgctgt cagaccaagt ttactcatat atactttaga 5341 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 5401 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 5461 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 5521 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 5581 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 5641 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 5701 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 5761 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 5821 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 5881 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 5941 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 6001 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 6061 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 6121 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 6181 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 6241 cggaagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 6301 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 6361 aagggcatcg gtcgacagca tctagttcga acacacaaaa aaccaacaca cagatgtaat 6421 gaaaataaag atattttatt gcggccatga ccggttctag atcattaggc gccaggtttt 6481 cgtgtcatac accaggtcct aggtccctcg ggcacctcga catcggcggt cacggtgaag 6541 cccaggcgct cgtagaatgg aaggtttctt ggtgcgcttg tttcgagaaa ggcgggcaca 6601 ccggcgcgtt cggcggcctc tacgccgggc aacacgacgg cgctgcccaa gcccttgccc 6661 tggtggtcgg ggctcactcc tacagtggcc agaaaccagg caggctcctt gggacgatgg 6721 ggggcaagca ggccctccat ttgctgctgg gcggccaggc ggctgccgct cagctcggcc 6781 attcgggggc cgatctcggc gaacacggcg ccggcctcaa cgctctcggg ggtagtccac 6841 acggccacgg ccgcgccgtc gtcggctacc cacaccttgc cgatgtccag cccgacgcgg 6901 gtcaggaaca gctcctgcaa ctctgtcaca cgctcgatgt gccggtcggg gtccacggtg 6961 tgccgggtag cggggtagtc ggcgaaagcg gcggccagag tgcggacggc gcggggcaca 7021 tcatcgcgag tggccaggcg cacggtaggc ttgtactcgg tcatggtggc gctagtgtca 7081 gaagaatcga gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg 7141 aatagctcag aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca 7201 tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc 7261 gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag 7321 cctggggact ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg 7381 cctgctgggg agcctgggga ctttccacac cctaactgac acacattcca cagctggttc 7441 tttccgcctc agaaggtagc taaccaagtt cctctttcag aggttatttc aggccatggt 7501 gctgcgcaga tcagcggaag agcgcccaat acgcaaacgg atcc //