LOCUS Exported 6532 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 6532) 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 6532) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6532 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(55..74) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 246..625 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 626..829 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 780..800 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 874..893 /label=T7 /note="T7 promoter, forward primer" promoter 874..892 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 933..980 /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 1257..1313 /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(2096..2114) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2097..2114) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2134..2151) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2140..2364 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2410..2838 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2497..2516) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2707..2728 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2847..2867) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2852..3181 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3032..3167 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3094..3113 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3248..4042 /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(3302..3321) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3912..3931 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4216..4337 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4253..4272) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4307..4326 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4386..4402) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4386..4402) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4399..4421) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4410..4426 /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(4434..4464) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4479..4500 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4617..4634) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4788..5376) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4868..4887) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5547..6407) /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 6170..6189 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6408..6512) /gene="bla" /label=AmpR promoter ORIGIN 1 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat 61 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc 121 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat 181 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac 241 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 301 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 361 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 421 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 481 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 541 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 601 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 661 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 721 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 781 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac 841 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca 901 agcttggtac cgagctcgga tccgccacca tgcgggacca catggtgctg cacgagtacg 961 tgaacgccgc tggcatcaca ggcggagatg gcggatctgg cggcggaagc aagctgagag 1021 tgttcctggc cctgcccatc atcatggtgg tggccttcag catgtgcatc atctgcctgc 1081 tgatggccgg cgacacctgg acagagacac tggcctacgt gctgttctgg ggcgtggcca 1141 gcatcggcac ctttttcatc atcctgtaca acggcaagga cttcgtggac gcccccagac 1201 tggtgcagaa agagaagatc gaccccgggc ttaaggagct cgcatgcgga agcggagcta 1261 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctagatctg 1321 aattcatggt gggccggaac agcgccatcg ccgcgggcgt gtgcggtgcc ctcttcatag 1381 ggtactgcat ctactttgac cgcaaaaggc ggagtgaccc caactccaaa ggagaagaac 1441 tgtttaccgg cgtggtgcca attctcgtgg aactggatgg cgatgtgaat ggccacaaat 1501 tttctgtcag aggagagggt gaaggtgatg ccacaatcgg aaagctcacc ctgaaattca 1561 tctgcaccac tggaaagctc cctgtgccat ggccaacact ggtcactacc ctgacctacg 1621 gcgtgcagtg cttttccaga tacccagacc atatgaagag gcatgacttt ttcaagagcg 1681 ccatgcccga gggctatgtg caggagagaa ccatctcttt caaagatgac gggaaataca 1741 agacccgcgc tgtggtcaag ttcgaaggag acacactggt gaatagaatc gagttgaagg 1801 gcacagactt taaggaagat ggaaacattc tcggccacaa gctggaatac aactttaact 1861 cccacaatgt gtacatcaca gccgacaagc aaaagaatgg catcaaggct aacttcacag 1921 tcagacacaa cgtcgaggat ggaagcgtgc agctggccga ccattatcaa cagaacactc 1981 caatcggcga cggccctgtg ctcctcccag acaaccatta cctgtccacc cagacagtcc 2041 tgagcaaaga tccaaatgaa aaaggaacat aactcgagcc tctagagggc cctattctat 2101 agtgtcacct aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc 2161 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg 2221 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc 2281 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg 2341 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg 2401 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2461 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 2521 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 2581 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 2641 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 2701 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2761 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 2821 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc 2881 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg 2941 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 3001 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc 3061 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc 3121 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa 3181 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc 3241 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 3301 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 3361 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 3421 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 3481 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 3541 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 3601 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3661 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3721 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 3781 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3841 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 3901 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3961 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 4021 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 4081 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 4141 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 4201 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc 4261 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 4321 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa 4381 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 4441 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 4501 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 4561 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4621 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4681 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4741 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4801 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4861 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4921 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4981 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 5041 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 5101 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 5161 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 5221 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5281 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 5341 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 5401 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 5461 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 5521 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5581 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5641 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5701 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5761 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5821 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5881 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 5941 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 6001 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 6061 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 6121 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 6181 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 6241 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 6301 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 6361 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 6421 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 6481 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gc //