LOCUS Exported 6533 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Detect the short Mitochondria-Endoplasmic reticulum contact . ACCESSION . VERSION . KEYWORDS SPLICS Mt-ER Short P2A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6533) AUTHORS Vallese F, Catoni C, Cieri D, Barazzuol L, Ramirez O, Calore V, Bonora M, Giamogante F, Pinton P, Brini M, Cali T TITLE An expanded palette of improved SPLICS reporters detects multiple organelle contacts in vitro and in vivo. JOURNAL Nat Commun. 2020 Nov 27;11(1):6069. doi: 10.1038/s41467-020-19892-6. PUBMED 33247103 REFERENCE 2 (bases 1 to 6533) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6533 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(44..63) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 235..614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 615..818 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 769..789 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 863..882 /label=T7 /note="T7 promoter, forward primer" promoter 863..881 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 922..969 /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" CDS 1246..1302 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" promoter complement(2085..2103) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2086..2103) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2123..2140) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2129..2353 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2399..2827 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2486..2505) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2696..2717 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2836..2856) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2841..3171 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3022..3157 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3084..3103 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3238..4032 /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(3292..3311) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3902..3921 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4206..4327 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4243..4262) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4297..4316 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4376..4392) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4376..4392) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4389..4411) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4400..4416 /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(4424..4454) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4469..4490 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4607..4624) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4778..5366) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4858..4877) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5537..6397) /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 6160..6179 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6398..6502) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 901 gagctcggat ccgccaccat gcgggaccac atggtgctgc acgagtacgt gaacgccgct 961 ggcatcacag gcggagatgg cggatctggc ggcggaagca agctgagagt gttcctggcc 1021 ctgcccatca tcatggtggt ggccttcagc atgtgcatca tctgcctgct gatggccggc 1081 gacacctgga cagagacact ggcctacgtg ctgttctggg gcgtggccag catcggcacc 1141 tttttcatca tcctgtacaa cggcaaggac ttcgtggacg cccccagact ggtgcagaaa 1201 gagaagatcg accccgggct taaggagctc gcatgcggaa gcggagctac taacttcagc 1261 ctgctgaagc aggctggaga cgtggaggag aaccctggac ctagatctga attcatggtg 1321 ggccggaaca gcgccatcgc cgcgggcgtg tgcggtgccc tcttcatagg gtactgcatc 1381 tactttgacc gcaaaaggcg gagtgacccc aactccaaag gagaagaact gtttaccggc 1441 gtggtgccaa ttctcgtgga actggatggc gatgtgaatg gccacaaatt ttctgtcaga 1501 ggagagggtg aaggtgatgc cacaatcgga aagctcaccc tgaaattcat ctgcaccact 1561 ggaaagctcc ctgtgccatg gccaacactg gtcactaccc tgacctacgg cgtgcagtgc 1621 ttttccagat acccagacca tatgaagagg catgactttt tcaagagcgc catgcccgag 1681 ggctatgtgc aggagagaac catctctttc aaagatgacg ggaaatacaa gacccgcgct 1741 gtggtcaagt tcgaaggaga cacactggtg aatagaatcg agttgaaggg cacagacttt 1801 aaggaagatg gaaacattct cggccacaag ctggaataca actttaactc ccacaatgtg 1861 tacatcacag ccgacaagca aaagaatggc atcaaggcta acttcacagt cagacacaac 1921 gtcgaggatg gaagcgtgca gctggccgac cattatcaac agaacactcc aatcggcgac 1981 ggccctgtgc tcctcccaga caaccattac ctgtccaccc agacagtcct gagcaaagat 2041 ccaaatgaaa aaggaacata actcgagcct ctagagggcc ctattctata gtgtcaccta 2101 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 2161 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 2221 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 2281 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 2341 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 2401 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 2461 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 2521 ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt 2581 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 2641 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 2701 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 2761 gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 2821 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 2881 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 2941 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 3001 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 3061 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 3121 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 3181 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 3241 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 3301 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 3361 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 3421 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3481 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 3541 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 3601 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 3661 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 3721 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 3781 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 3841 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 3901 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 3961 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 4021 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 4081 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 4141 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 4201 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 4261 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 4321 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 4381 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 4441 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 4501 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4561 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4621 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4681 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4741 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4801 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4861 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4921 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac 4981 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5041 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5101 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5161 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 5221 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5281 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5341 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5401 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 5461 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 5521 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 5581 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 5641 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 5701 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 5761 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 5821 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 5881 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 5941 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 6001 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 6061 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 6121 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 6181 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6241 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6301 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 6361 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 6421 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 6481 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc //