LOCUS Exported 6883 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Detect the long Mitochondria-Endoplasmic reticulum contact . ACCESSION . VERSION . KEYWORDS SPLICS Mt-ER Long P2A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6883) 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 6883) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6883 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(32..51) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 223..602 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 603..806 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 757..777 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 851..870 /label=T7 /note="T7 promoter, forward primer" promoter 851..869 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 910..957 /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 994..1260 /codon_start=1 /product="FKBP-rapamycin binding domain of human FRAP" /label=FRB /translation="MWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF NQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ" CDS 1585..1641 /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(2424..2442) /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind complement(2425..2442) /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(2462..2479) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2468..2692 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 2738..3166 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2825..2844) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3035..3056 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3175..3195) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3180..3509 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 3360..3495 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3422..3441 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 3576..4370 /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(3630..3649) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 4240..4259 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 4544..4665 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4581..4600) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4635..4654 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4714..4730) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4714..4730) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4727..4749) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4738..4754 /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(4762..4792) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4807..4828 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4945..4962) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5116..5704) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5196..5215) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5875..6735) /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 6498..6517 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6736..6840) /gene="bla" /label=AmpR promoter ORIGIN 1 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 61 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 121 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 181 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 241 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 301 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 361 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 421 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 481 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 541 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca 601 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat 661 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 721 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac 781 ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact gcttactggc 841 ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga gctcggatcc 901 gccaccatgc gggaccacat ggtgctgcac gagtacgtga acgccgctgg catcacaggc 961 ggagatggcg gatctggcgg cggaagcaaa ctgatgtggc acgagggact ggaagaggcc 1021 agcagactgt acttcggcga gcggaacgtg aagggcatgt tcgaggtgct ggaacccctg 1081 cacgccatga tggaaagagg cccccagacc ctgaaagaga caagcttcaa ccaggcctac 1141 ggccgggacc tgatggaagc ccaggaatgg tgccggaagt acatgaagtc cggcaatgtg 1201 aaggacctga cacaggcctg ggacctgtac taccacgtgt tccggcggat cagcaagcag 1261 ggctctgaag ccgccgctag agaagctgct gctagaggcg gagcttctgg cgctggcgca 1321 ggcgctgggg ccatcctgaa tagcagagtg ttcctggccc tgcccatcat catggtggtg 1381 gccttcagca tgtgcatcat ctgcctgctg atggccggcg acacctggac agagacactg 1441 gcctacgtgc tgttctgggg cgtggccagc atcggcacct ttttcatcat cctgtacaac 1501 ggcaaggact tcgtggacgc ccccaggctg gtgcagaaag agaagatcga ccccgggctt 1561 aaggagctcg catgcggaag cggagctact aacttcagcc tgctgaagca ggctggagac 1621 gtggaggaga accctggacc tagatctgaa ttcatggtgg gccggaacag cgccatcgcc 1681 gcgggcgtgt gcggtgccct cttcataggg tactgcatct actttgaccg caaaaggcgg 1741 agtgacccca actccaaagg agaagaactg tttaccggcg tggtgccaat tctcgtggaa 1801 ctggatggcg atgtgaatgg ccacaaattt tctgtcagag gagagggtga aggtgatgcc 1861 acaatcggaa agctcaccct gaaattcatc tgcaccactg gaaagctccc tgtgccatgg 1921 ccaacactgg tcactaccct gacctacggc gtgcagtgct tttccagata cccagaccat 1981 atgaagaggc atgacttttt caagagcgcc atgcccgagg gctatgtgca ggagagaacc 2041 atctctttca aagatgacgg gaaatacaag acccgcgctg tggtcaagtt cgaaggagac 2101 acactggtga atagaatcga gttgaagggc acagacttta aggaagatgg aaacattctc 2161 ggccacaagc tggaatacaa ctttaactcc cacaatgtgt acatcacagc cgacaagcaa 2221 aagaatggca tcaaggctaa cttcacagtc agacacaacg tcgaggatgg aagcgtgcag 2281 ctggccgacc attatcaaca gaacactcca atcggcgacg gccctgtgct cctcccagac 2341 aaccattacc tgtccaccca gacagtcctg agcaaagatc caaatgaaaa aggaacataa 2401 ctcgagcctc tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca 2461 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2521 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2581 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2641 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 2701 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 2761 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 2821 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 2881 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 2941 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 3001 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 3061 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 3121 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 3181 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 3241 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 3301 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 3361 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 3421 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 3481 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 3541 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 3601 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 3661 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 3721 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 3781 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 3841 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 3901 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 3961 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 4021 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 4081 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 4141 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 4201 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 4261 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 4321 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 4381 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 4441 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 4501 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 4561 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 4621 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 4681 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 4741 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 4801 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 4861 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 4921 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 4981 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 5041 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 5101 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 5161 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 5221 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 5281 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 5341 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 5401 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 5461 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 5521 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 5581 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 5641 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 5701 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 5761 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 5821 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 5881 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 5941 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 6001 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 6061 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 6121 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 6181 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 6241 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 6301 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 6361 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 6421 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 6481 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 6541 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 6601 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 6661 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 6721 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 6781 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 6841 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg gga //