LOCUS Exported 7799 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION N-terminal 3xFLAG tag and C-terminal EGFP tag. Gateway destination vector for mammalian expression.. ACCESSION . VERSION . KEYWORDS pDEST-CMV-3xFLAG-gateway-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7799) 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 7799) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7799 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(46..65) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 237..616 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 617..820 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 771..791 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 857..922 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" protein_bind 926..1050 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 1075..1105 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 1159..1839 /codon_start=1 /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE DPAY" primer_bind complement(1225..1244) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 2159..2464 /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 2343..2362 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(2505..2629) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 2646..3365 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 2646..3362 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2691..2712) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2952..2971) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3299..3320 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(3392..3409) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3398..3622 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 3668..4096 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3755..3774) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3965..3986 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4105..4125) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4110..4439 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4290..4425 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4352..4371 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 4506..5300 /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(4560..4579) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5170..5189 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 5474..5595 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5511..5530) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 5565..5584 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(5644..5660) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5644..5660) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5657..5679) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5668..5684 /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(5692..5722) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5737..5758 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5875..5892) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6046..6634) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6126..6145) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6805..7665) /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 7428..7447 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7666..7770) /gene="bla" /label=AmpR promoter ORIGIN 1 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga 61 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg 121 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct 181 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca 241 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 301 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 361 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 421 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 481 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 541 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 601 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 661 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 721 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 781 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagaa 841 ttgatctggt accatggact acaaagacca tgacggtgat tataaagatc atgacatcga 901 ttacaaggat gacgatgaca agcttacaag tttgtacaaa aaagctgaac gagaaacgta 961 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata 1021 ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca 1081 ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca 1141 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc 1201 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac 1261 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag 1321 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt 1381 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt 1441 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg 1501 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc 1561 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc 1621 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc 1681 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc 1741 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag 1801 tggcaggcgg ggcgtaatct agaggatccg gcttactaaa agccagataa cagtatgcgt 1861 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt 1921 caaaaagagg tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca 1981 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat 2041 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag 2101 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacagggg ctggtgaaat 2161 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca 2221 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct 2281 gctgtcagat aaagtccccc gtgaacttta cccggtggtg catatcgggg atgaaagctg 2341 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc 2401 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat 2461 ataaatgtca ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat 2521 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga 2581 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtt cgatctagac 2641 tcgagatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 2701 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 2761 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 2821 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 2881 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 2941 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 3001 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 3061 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 3121 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 3181 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 3241 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 3301 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 3361 agtaattaat taagataaac ccgctgatca gcctcgactg tgccttctag ttgccagcca 3421 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3481 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3541 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3601 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg 3661 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 3721 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 3781 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 3841 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 3901 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 3961 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 4021 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 4081 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 4141 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 4201 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 4261 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 4321 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg 4381 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 4441 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt 4501 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 4561 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 4621 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 4681 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 4741 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 4801 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 4861 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 4921 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 4981 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 5041 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 5101 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 5161 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 5221 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 5281 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc 5341 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 5401 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 5461 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 5521 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 5581 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 5641 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 5701 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 5761 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 5821 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 5881 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5941 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 6001 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6061 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 6121 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6181 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6241 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6301 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6361 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6421 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6481 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6541 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6601 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6661 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6721 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6781 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 6841 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 6901 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 6961 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 7021 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 7081 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 7141 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 7201 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7261 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7321 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7381 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7441 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7501 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7561 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7621 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 7681 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 7741 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacg //