LOCUS Exported 6136 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression of rat LC3 fused to mRFP and EGFP. ACCESSION . VERSION . KEYWORDS ptfLC3 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6136) AUTHORS Kimura S, Noda T, Yoshimori T TITLE Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. JOURNAL Autophagy. . 3(5):452-60. PUBMED 17534139 REFERENCE 2 (bases 1 to 6136) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6136 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 108..411 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 412..615 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 566..586 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 645..654 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 651..1325 /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /note="mammalian codon-optimized" /translation="MAFSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS TGA" primer_bind complement(781..799) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1049..1068 /label=mCherry-F /note="mCherry, forward primer" primer_bind 1243..1266 /label=DsRed1-C /note="DsRed1, forward primer" regulatory 1338..1347 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1344..2060 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 1344..2060 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1389..1410) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1650..1669) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1997..2018 /label=EGFP-C /note="EGFP, forward primer" CDS 2079..2501 /codon_start=1 /gene="Map1lc3b" /product="rat LC3 protein" /label=LC3 /note="Atg8 family member found on autophagosomes" /translation="PSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPV LDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDE DGFLYMVYASQETFGTALAVTYMSALKATATGREPCL" polyA_signal 2971..3092 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3008..3027) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3062..3081 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin complement(3099..3554) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3236..3257) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3448..3467 /label=F1ori-R /note="F1 origin, reverse primer" promoter 3581..3685 /gene="bla" /label=AmpR promoter promoter 3687..4044 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(3710..3730) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 3895..4030 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3957..3976 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4079..4873 /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(4133..4152) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4743..4762 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(5061..5080) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5105..5152 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 5481..6069 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5970..5989 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag 61 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 121 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 181 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 241 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 301 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 361 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg 421 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc 481 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa 541 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 601 tatataagca gagctggttt agtgaaccgt cagatccgct agcggccacc atggccttct 661 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg 721 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 781 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc 841 agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 901 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 961 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc 1021 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 1081 aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga 1141 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca 1201 agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc 1261 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg 1321 gcgccggtgg accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg 1381 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 1441 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 1501 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 1561 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 1621 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 1681 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 1741 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 1801 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 1861 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 1921 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 1981 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 2041 gcatggacga gctgtacaag tccggactca gatctagacc gtccgagaag accttcaaac 2101 agcgccggag cttcgaacaa agagtggaag atgtccggct catccgggag cagcacccca 2161 ccaagatccc agtgattata gagcgataca agggtgagaa gcagctgccc gtcctggaca 2221 agaccaagtt ccttgtacct gatcacgtga atatgagcga actcatcaag ataattagaa 2281 ggcgcctgca gctcaatgct aaccaagcct tcttcctcct ggtgaatggg cacagcatgg 2341 tgagtgtgtc cacacccatc tctgaagtgt acgagagcga gagagatgaa gacggcttcc 2401 tgtacatggt ctatgcctcc caggagacgt tcgggacagc actggctgtt acatacatgt 2461 cagctctgaa ggcaacagca acaggaagag agccatgctt gtgacagaca tacagccact 2521 tccaactaaa gcaagcctct gctccctgct acctgcatgg agcccactgt gacactcaga 2581 ccatccccgg tcactcactc actcgtgtct gagaatctca gtgagagctg cctctgtcac 2641 ggacggaagc caacacagcc acctctcgac ctggctcccc acagcaccca ccctccctgc 2701 atgcagctgt ccctgctaac ccccaatgtt atgttacact gtgtaagtcc ccactgctgc 2761 cgtgtgtggg ttgtgtacgt cgtcatgtcc ctggtttata actatggtgc gaattctgca 2821 gtcgacggta ccgcgggccc gggatccacc ggatctagat aactgatcat aatcagccat 2881 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg 2941 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac 3001 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 3061 tgtggtttgt ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt 3121 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 3181 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt 3241 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 3301 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag 3361 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 3421 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 3481 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc 3541 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 3601 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 3661 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt 3721 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 3781 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg 3841 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg 3901 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt 3961 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt 4021 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat 4081 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 4141 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 4201 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 4261 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 4321 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 4381 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 4441 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 4501 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 4561 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 4621 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 4681 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 4741 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 4801 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 4861 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 4921 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 4981 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 5041 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct 5101 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc 5161 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa 5221 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg 5281 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt 5341 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 5401 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 5461 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 5521 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 5581 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 5641 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 5701 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 5761 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 5821 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 5881 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 5941 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 6001 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6061 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6121 gctcacatgt tctttc //