LOCUS Exported 7071 bp ds-DNA circular SYN 20-JAN-2022 DEFINITION ER protein alpha-1-antitrypsin containing E342K mutation (ATZ mutant) fused to blue fluorescence protein (BFP) tag protein, an HA tag and the eleventh beta-strand of GFP. ACCESSION . VERSION . KEYWORDS pATZ-BFP-HA-GFP11 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7071) TITLE ER-misfolded proteins become sequestered with mitochondria and impair mitochondrial function JOURNAL Unpublished REFERENCE 2 (bases 1 to 7071) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 20, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7071 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 66..423 /label=SV40 promoter /note="SV40 enhancer and early promoter" primer_bind complement(89..109) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin 274..409 /label=SV40 ori /note="SV40 origin of replication" primer_bind 336..355 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 454..1491 /codon_start=1 /product="hygromycin B phosphotransferase" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK EVGRV" RBS 1470..1478 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" polyA_signal 1515..1563 /note="synthetic polyadenylation signal" primer_bind complement(1719..1736) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1890..2478) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1970..1989) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2678..3538) /codon_start=1 /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" polyA_signal 3643..3691 /note="synthetic polyadenylation signal" misc_feature 3705..3796 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 3745..3764 /label=RVprimer3 /note="pGL3 vector, forward primer" enhancer 3947..4326 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 4327..4530 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 4481..4501 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 4527..4551 /label=LNCX /note="Human CMV promoter, forward primer" CDS 5852..6550 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC DLPSKLGHKLN" misc_feature 6551..6616 /label=MCS /note="multiple cloning site" CDS 6662..6688 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 6728..6775 /codon_start=1 /product="11th beta-strand of superfolder GFP" /label=GFP11 /note="associates with GFP(1-10), consisting of the first 10 beta-strands, to reconstitute fluorescent GFP (Cabantous et al., 2005; Kamiyama et al., 2016)" /translation="RDHMVLHEYVNAAGIT" polyA_signal 6822..6943 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6833..6852) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 6887..6906 /label=SV40pA-R /note="SV40 polyA, reverse primer" ORIGIN 1 ccgctgatct gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttagct 61 accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc 121 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg 181 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 241 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 301 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 361 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc 421 aaaaagctcg attcttctga cactagcgcc accatgaaga agcccgaact caccgctacc 481 agcgttgaaa aatttctcat cgagaagttc gacagtgtga gcgacctgat gcagttgtcg 541 gagggcgaag agagccgagc cttcagcttc gatgtcggcg gacgcggcta tgtactgcgg 601 gtgaatagct gcgctgatgg cttctacaaa gaccgctacg tgtaccgcca cttcgccagc 661 gctgcactac ccatccccga agtgttggac atcggcgagt tcagcgagag cctgacatac 721 tgcatcagta gacgcgccca aggcgttact ctccaagacc tccccgaaac agagctgcct 781 gctgtgttac agcctgtcgc cgaagctatg gatgctattg ccgccgccga cctcagtcaa 841 accagcggct tcggcccatt cgggccccaa ggcatcggcc agtacacaac ctggcgggat 901 ttcatttgcg ccattgctga tccccatgtc taccactggc agaccgtgat ggacgacacc 961 gtgtccgcca gcgtagctca agccctggac gaactgatgc tgtgggccga agactgtccc 1021 gaggtgcgcc acctcgtcca tgccgacttc ggcagcaaca acgtcctgac cgacaacggc 1081 cgcatcaccg ccgtaatcga ctggtccgaa gctatgttcg gggacagtca gtacgaggtg 1141 gccaacatct tcttctggcg gccctggctg gcttgcatgg agcagcagac tcgctacttc 1201 gagcgccggc atcccgagct ggccggcagc cctcgtctgc gagcctacat gctgcgcatc 1261 ggcctggatc agctctacca gagcctcgtg gacggcaact tcgacgatgc tgcctgggct 1321 caaggccgct gcgatgccat cgtccgcagc ggggccggca ccgtcggtcg cacacaaatc 1381 gctcgccgga gcgcagccgt atggaccgac ggctgcgtcg aggtgctggc cgacagcggc 1441 aaccgccggc ccagtacacg accgcgcgct aaggaggtag gtcgagttta aactctagaa 1501 ccggtcatgg ccgcaataaa atatctttat tttcattaca tctgtgtgtt ggttttttgt 1561 gtgttcgaac tagatgctgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct 1621 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc 1681 aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc 1741 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 1801 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 1861 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 1921 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 1981 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 2041 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 2101 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 2161 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 2221 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2281 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 2341 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2401 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 2461 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 2521 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 2581 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2641 tctgacagcg gccgcaaatg ctaaaccact gcagtggtta ccagtgcttg atcagtgagg 2701 caccgatctc agcgatctgc ctatttcgtt cgtccatagt ggcctgactc cccgtcgtgt 2761 agatcactac gattcgtgag ggcttaccat caggccccag cgcagcaatg atgccgcgag 2821 agccgcgttc accggccccc gatttgtcag caatgaacca gccagcaggg agggccgagc 2881 gaagaagtgg tcctgctact ttgtccgcct ccatccagtc tatgagctgc tgtcgtgatg 2941 ctagagtaag aagttcgcca gtgagtagtt tccgaagagt tgtggccatt gctactggca 3001 tcgtggtatc acgctcgtcg ttcggtatgg cttcgttcaa ctctggttcc cagcggtcaa 3061 gccgggtcac atgatcaccc atattatgaa gaaatgcagt cagctcctta gggcctccga 3121 tcgttgtcag aagtaagttg gccgcggtgt tgtcgctcat ggtaatggca gcactacaca 3181 attctcttac cgtcatgcca tccgtaagat gcttttccgt gaccggcgag tactcaacca 3241 agtcgttttg tgagtagtgt atacggcgac caagctgctc ttgcccggcg tctatacggg 3301 acaacaccgc gccacatagc agtactttga aagtgctcat catcgggaat cgttcttcgg 3361 ggcggaaaga ctcaaggatc ttgccgctat tgagatccag ttcgatatag cccactcttg 3421 cacccagttg atcttcagca tcttttactt tcaccagcgt ttcggggtgt gcaaaaacag 3481 gcaagcaaaa tgccgcaaag aagggaatga gtgcgacacg aaaatgttgg atgctcatac 3541 tcgtcctttt tcaatattat tgaagcattt atcagggtta ctagtacgtc tctcaaggat 3601 aagtaagtaa tattaaggta cgggaggtat tggacaggcc gcaataaaat atctttattt 3661 tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata cgctctccat 3721 caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc aagtgcaggt 3781 gccagaacat ttctctggcc taactggcct caatattggc cattagccat attattcatt 3841 ggttatatag cataaatcaa tattggctat tggccattgc atacgttgta tctatatcat 3901 aatatgtaca tttatattgg ctcatgtcca atatgaccgc catgttggca ttgattattg 3961 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc 4021 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 4081 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 4141 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 4201 ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 4261 tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 4321 accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt tgactcacgg 4381 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa 4441 cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg cggtaggcgt 4501 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cactagaagc 4561 ttcgatgccg tcttctgtct cgtggggcat cctcctgctg gcaggcctgt gctgcctggt 4621 ccctgtctcc ctggctgagg atccccaggg agatgctgcc cagaagacag atacatccca 4681 ccatgatcag gatcacccaa ccttcaacaa gatcaccccc aacctggctg agttcgcctt 4741 cagcctatac cgccagctgg cacaccagtc caacagcacc aatatcttct tctccccagt 4801 gagcatcgct acagcctttg caatgctctc cctggggacc aaggctgaca ctcacgatga 4861 aatcctggag ggcctgaatt tcaacctcac ggagattccg gaggctcaga tccatgaagg 4921 cttccaggaa ctcctccgta ccctcaacca gccagacagc cagctccagc tgaccaccgg 4981 caatggcctg ttcctcagcg agggcctgaa gctagtggat aagtttttgg aggatgttaa 5041 aaagttgtac cactcagaag ccttcactgt caacttcggg gacaccgaag aggccaagaa 5101 acagatcaac gattacgtgg agaagggtac tcaagggaaa attgtggatt tggtcaagga 5161 gcttgacaga gacacagttt ttgctctggt gaattacatc ttctttaaag gcaaatggga 5221 gagacccttt gaagtcaagg acaccgagga agaggacttc cacgtggacc aggtgaccac 5281 cgtgaaggtg cctatgatga agcgtttagg catgtttaac atccagcact gtaagaagct 5341 gtccagctgg gtgctgctga tgaaatacct gggcaatgcc accgccatct tcttcctgcc 5401 tgatgagggg aaactacagc acctggaaaa tgaactcacc cacgatatca tcaccaagtt 5461 cctggaaaat gaagacagaa ggtctgccag cttacattta cccaaactgt ccattactgg 5521 aacctatgat ctgaagagcg tcctgggtca actgggcatc actaaggtct tcagcaatgg 5581 ggctgacctc tccggggtca cagaggaggc acccctgaag ctctccaagg ccgtgcataa 5641 ggctgtgctg accatcgaca agaaagggac tgaagctgct ggggccatgt ttttagaggc 5701 catacccatg tctatccccc ccgaggtcaa gttcaacaaa ccctttgtct tcttaatgat 5761 tgaacaaaat accaagtctc ccctcttcat gggaaaagtg gtgaatccca cccaaaaacc 5821 cgggggatcg ggtgacggtc cggtcgccac catgagcgag ctgattaagg agaacatgca 5881 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg 5941 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc 6001 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat 6061 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg 6121 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct 6181 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg 6241 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc 6301 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct 6361 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc 6421 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta 6481 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca 6541 caagcttaat tccggactca gatctcgagc tcaagcttcg aattctgcag tcgacggtac 6601 cgcgggcccg ggatccaccg gatctagacc cgggggatcg ggtgacggtc cggtcgccac 6661 ctacccatac gatgttccag attacgctgg tcgacggatc ggtggtggtt ctggaggcgg 6721 ttctatgaga gatcatatgg ttttgcatga atatgttaat gctgctggta ttacttaata 6781 attctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt 6841 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc 6901 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat 6961 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct 7021 ctacaaatgt ggtaaaatcg ataaggatcc gtttgcgtat tgggcgctct t //