LOCUS Exported 5118 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses 3xFLAG-LC3B G120A in mammalian cells.. ACCESSION . VERSION . KEYWORDS pCMV 3xFLAG-LC3B G120A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5118) AUTHORS Agrotis A, Pengo N, Burden JJ, Ketteler R TITLE Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells. JOURNAL Autophagy. 2019 Jan 21. doi: 10.1080/15548627.2019.1569925. PUBMED 30661429 REFERENCE 2 (bases 1 to 5118) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5118 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(21..609) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(101..120) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(780..1640) /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 1403..1422 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1641..1745) /gene="bla" /label=AmpR promoter rep_origin 1772..2227 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1859..1878) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2069..2090 /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2353..2375 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2367..2384 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2368..2384 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" enhancer 2545..2924 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 2925..3128 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 3079..3099 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 3165..3230 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" protein_bind 3234..3258 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 3261..3635 /codon_start=1 /gene="MAP1LC3B" /product="human LC3 protein" /label=LC3B /note="Atg8 family member found on autophagosomes" /translation="MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKD EDGFLYMVYASQETFAMKLSV" protein_bind complement(3640..3664) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" polyA_signal 3682..4304 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3881..3900) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" primer_bind complement(4328..4348) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4333..4662 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4513..4648 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4575..4594 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4704..4723) /label=T7 /note="T7 promoter, forward primer" promoter complement(4705..4723) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4737..4753) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4737..4753) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4750..4772) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4761..4777 /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(4785..4815) /label=lac promoter /note="promoter for the E. coli lac operon" primer_bind complement(4968..4985) /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 61 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 121 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 181 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 241 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 301 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 361 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 421 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 481 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 541 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 601 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 661 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 721 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 781 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 841 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 901 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 961 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 1021 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 1081 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 1141 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 1201 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 1261 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 1321 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 1381 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 1441 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 1501 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 1561 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 1621 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 1681 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 1741 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg 1801 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 1861 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 1921 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 1981 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 2041 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 2101 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 2161 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt 2221 acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 2281 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa 2341 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag ctgatctata 2401 cattgaatca atattggcaa ttagccatat tagtcattgg ttatatagca taaatcaata 2461 ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct 2521 catgtccaat atgaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa 2581 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 2641 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2701 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2761 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg 2821 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc 2881 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2941 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 3001 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 3061 ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 3121 gcagagctcg tttagtgaac cgtcagaatt gatctggtac catggactac aaagaccatg 3181 acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag cttacaagtt 3241 tgtacaaaaa agcaggctta atgccgtcgg agaagacctt caagcagcgg cgcaccttcg 3301 aacaaagagt agaagatgtc cgacttattc gagagcagca tccaaccaaa atcccggtga 3361 taatagaacg atacaagggt gagaagcagc ttcctgttct ggataaaaca aagttccttg 3421 tacctgacca tgtcaacatg agtgagctca tcaagataat tagaaggcgc ttacagctca 3481 atgctaatca ggccttcttc ctgttggtga acggacacag catggtcagc gtctccacac 3541 caatctcaga ggtgtatgag agtgagaaag atgaagatgg attcctgtac atggtctatg 3601 cctcccagga gacgttcgcg atgaaattgt cagtgtaata cccagctttc ttgtacaaag 3661 tggtgtgatc tagaggatcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg 3721 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc 3781 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc 3841 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg 3901 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc 3961 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt 4021 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa 4081 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact 4141 gctcccttcc ctgtccttct gattttaaaa taactatacc agcaggagga cgtccagaca 4201 cagcataggc tacctggcca tgcccaaccg gtgggacatt tgagttgctt gcttggcact 4261 gtcctctcat gcgttgggtc cactcagtag atgcctgttg aattgggtac gcggccagct 4321 tggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 4381 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 4441 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 4501 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 4561 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag 4621 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcct cgatcgagga 4681 actgaaaaac cagaaagtta attccctata gtgagtcgta ttaaattcgt aatcatgtca 4741 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 4801 agcataaagt gtaaagcctg gggtgcctaa tgagcgagct aactcacatt aattgcgttg 4861 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 4921 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 4981 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5041 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5101 aaggccagga accgtaaa //