LOCUS Exported 8341 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expression of GFP targeted to mitochondria with Flag and HA tags. ACCESSION . VERSION . KEYWORDS pLYS1-FLAG-MitoGFP-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8341) AUTHORS Sancak Y, Markhard AL, Kitami T, Kovacs-Bogdan E, Kamer KJ, Udeshi ND, Carr SA, Chaudhuri D, Clapham DE, Li AA, Calvo SE, Goldberger O, Mootha VK TITLE EMRE is an Essential Component of the Mitochondrial Calcium Uniporter Complex. JOURNAL Science. 2013 Nov 14. PUBMED 24231807 REFERENCE 2 (bases 1 to 8341) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8341 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 131..334 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 285..305 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 549..572 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 603..1319 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 603..1319 /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(648..669) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(909..928) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1256..1277 /label=EGFP-C /note="EGFP, forward primer" CDS 1347..1373 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" misc_feature 1424..1541 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1590..2100 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2019..2037 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 2122..2721 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(2122..2141) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 2618..2638 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 2849..3082 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3154..3275 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3191..3210) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3245..3264 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3315..3450 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3377..3396 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3470..3489) /label=T7 /note="T7 promoter, forward primer" promoter complement(3471..3489) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3499..3516) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3499..3515) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3508..3530) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3657..4112 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3744..3763) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3954..3975 /label=F1ori-F /note="F1 origin, forward primer" promoter 4138..4242 /gene="bla" /label=AmpR promoter CDS 4243..5103 /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 complement(4461..4480) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5274..5862 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5763..5782 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6016..6033 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6150..6171 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6186..6216 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6224..6240 /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)." primer_bind 6229..6251 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6248..6264 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6248..6264 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6283..6303 /label=T3 /note="T3 promoter, forward primer" promoter 6285..6303 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6331..6557 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6558..6738 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6785..6910 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7403..7636 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7821..7865 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" primer_bind 8163..8183 /label=hU6-F /note="Human U6 promoter, forward primer" ORIGIN 1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta 361 gcatggcggc cgccgcaggt agatcgctcc tgctgctcct ctcctctcgg ggcggcggcg 421 gcgggggcgc cggcggctgc ggggcgctga ctgccggctg cttccctggg ctgggcgtca 481 gccgccaccg gcagcagcag caccaccgga cggtacacca gaggatcgct tccggaggca 541 ccggtatgga ttacaaggat gacgatgaca agggtggatc tggtggatct ggtggacccg 601 ggatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 661 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 721 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 781 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 841 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 901 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 961 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 1021 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 1081 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 1141 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 1201 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 1261 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg 1321 gtggatctgg tggatctggt ggatcttacc cctatgatgt gcctgactac gcataaggta 1381 ccgaattctc gacctcgaga caaatggcag tattcatcca caattttaaa agaaaagggg 1441 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa 1501 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca 1561 gcagagatcc actttggccg cggctcgagg gggttggggt tgcgcctttt ccaaggcagc 1621 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc 1681 gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct 1741 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg 1801 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag taccctcgca 1861 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc 1921 ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg 1981 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct 2041 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag 2101 ggggatccac cggagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc 2161 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg 2221 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc 2281 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg 2341 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg 2401 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg 2461 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac 2521 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc 2581 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg 2641 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg 2701 acccgcaagc ccggtgcctg acgcccgccc cacgacccgc agcgcccgac cgaaaggagc 2761 gcacgacccc atgcatcggt acctttaaga ccaatgactt acaaggcagc tgtagatctt 2821 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa 2881 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc 2941 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 3001 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag 3061 tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca gtatttataa 3121 cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt 3181 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 3241 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc 3301 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 3361 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 3421 agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga 3481 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 3541 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 3601 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc 3661 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 3721 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 3781 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 3841 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 3901 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 3961 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 4021 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 4081 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc 4141 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 4201 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 4261 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 4321 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4381 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4441 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4501 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4561 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4621 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4681 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4741 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4801 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 4861 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 4921 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 4981 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 5041 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 5101 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 5161 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 5221 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 5281 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 5341 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5401 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 5461 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5521 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5581 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5641 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 5701 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5761 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 5821 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 5881 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 5941 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 6001 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 6061 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 6121 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 6181 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 6241 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 6301 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt 6361 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat 6421 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc 6481 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc 6541 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg 6601 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 6661 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 6721 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga 6781 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg 6841 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg 6901 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag 6961 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga 7021 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg 7081 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt 7141 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga 7201 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct 7261 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag 7321 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga 7381 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa 7441 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta 7501 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac 7561 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa 7621 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg 7681 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct 7741 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag 7801 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa 7861 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga 7921 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag 7981 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg 8041 acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat 8101 tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggcc gcccccttca 8161 ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 8221 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 8281 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 8341 a //