LOCUS Exported 6452 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses GFP-tagged deconjugation-resistant LC3B in mammalian cells. ACCESSION . VERSION . KEYWORDS pDEST-CMV GFP-LC3B Q116P G120 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6452) AUTHORS Agrotis A, von Chamier L, Oliver H, Kiso K, Singh T, Ketteler R TITLE Human ATG4 autophagy proteases counteract attachment of ubiquitin-like LC3/GABARAP proteins to other cellular proteins. JOURNAL J Biol Chem. 2019 Jul 17. pii: AC119.009977. doi: 10.1074/jbc.AC119.009977. PUBMED 31315929 REFERENCE 2 (bases 1 to 6452) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6452 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 145..524 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 525..728 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 679..699 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 762..771 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 768..1484 /codon_start=1 /product="green fluorescent protein" /label=GFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITHGMDELYK" primer_bind complement(813..834) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1074..1093) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1421..1442 /label=EGFP-C /note="EGFP, forward primer" protein_bind 1506..1530 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" protein_bind complement(1897..1921) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(1953..1970) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 1959..2183 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2229..2657 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2316..2335) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2526..2547 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2666..2686) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2671..3000 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2851..2986 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2913..2932 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3067..3861 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3121..3140) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3731..3750 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4035..4156 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4072..4091) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4126..4145 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4205..4221) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4205..4221) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4218..4240) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4229..4245 /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)." promoter complement(4253..4283) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4298..4319 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4436..4453) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4607..5195) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4687..4706) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5366..6226) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5989..6008 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6227..6331) /gene="bla" /label=AmpR promoter primer_bind complement(6406..6425) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag 61 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg 121 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 541 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 601 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 661 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 721 gcagagctcg tttagtgaac cgtcagaatt gatctggtac cgccaccatg gtgagcaagg 781 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 841 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 901 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 961 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 1021 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 1081 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 1141 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 1201 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1261 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 1321 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 1381 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 1441 tgaccgccgc cgggatcact cacggcatgg acgagctcta caaaagcggt tccggaccgg 1501 atcaaacaag tttgtacaaa aaagcaggct taatgccgtc ggagaagacc ttcaagcagc 1561 ggcgcacctt cgaacaaaga gtagaagatg tccgacttat tcgagagcag catccaacca 1621 aaatcccggt gataatagaa cgatacaagg gtgagaagca gcttcctgtt ctggataaaa 1681 caaagttcct tgtacctgac catgtcaaca tgagtgagct catcaagata attagaaggc 1741 gcttacagct caatgctaat caggccttct tcctgttggt gaacggacac agcatggtca 1801 gcgtctccac accaatctca gaggtgtatg agagtgagaa agatgaagat ggattcctgt 1861 acatggtcta tgcctccccg gagacgttcg ggtaataccc agctttcttg tacaaagtgg 1921 tgataattaa ttaagataaa cccgctgatc agcctcgact gtgccttcta gttgccagcc 1981 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 2041 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 2101 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 2161 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 2221 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 2281 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 2341 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 2401 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 2461 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 2521 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 2581 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 2641 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 2701 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 2761 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 2821 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 2881 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct 2941 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 3001 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg 3061 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg 3121 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg 3181 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 3241 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca 3301 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg 3361 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat 3421 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa 3481 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg 3541 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg 3601 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg 3661 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat 3721 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac 3781 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 3841 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc 3901 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg 3961 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt 4021 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 4081 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 4141 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 4201 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 4261 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 4321 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 4381 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 4441 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4501 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4561 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4621 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4681 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4741 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4801 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4861 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4921 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4981 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5041 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5101 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5161 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5221 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5281 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5341 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5401 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5461 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5521 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5581 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 5641 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 5701 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 5761 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 5821 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 5881 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 5941 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6001 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6061 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6121 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6181 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6241 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6301 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 6361 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 6421 tgccgcatag ttaagccagt atctgctccc tg //