LOCUS       sequence-384083-        7389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of siRNA-resistant ORP8 tagged with mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7389)
  AUTHORS   Nguyen TT, Voeltz GK
  TITLE     An ER phospholipid hydrolase drives ER-associated mitochondrial 
            constriction for fission and fusion.
  JOURNAL   Elife. 2022 Nov 30;11:e84279. doi: 10.7554/eLife.84279.
  PUBMED    36448541
REFERENCE   2  (bases 1 to 7389)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-11-30"; volume: "11:e84279. doi"; pages: " 
            10.7554/eLife.84279"
COMMENT     Sequence Label: siRNA-resistant mNG-ORP8
FEATURES             Location/Qualifiers
     source          1..7389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1320
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      868..877
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4177..4298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4214..4233)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4268..4287
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4305..4760)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4442..4463)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4654..4673
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4787..4891
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4893..5250
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4893..5084
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4916..4936)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5101..5236
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5285..6079
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5285..6079
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5339..5358)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5949..5968
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6267..6286)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6311..6358
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6687..7275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7176..7195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca
      661 catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc
      721 accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc
      781 cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc
      841 taccctgacg ggatgtcgcc tttccaggcc gccatggtag atggctccgg ctaccaagtc
      901 catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac
      961 gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt
     1021 cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc
     1081 aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc
     1141 taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg
     1201 aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc
     1261 aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag
     1321 tccggactca gatctcgagc tcaagcttcg aattctgcag tcgacatgga gggaggtttg
     1381 gcagatggag aacctgatcg aacttcgctt cttggtgata gcaaagatgt ccttgggcca
     1441 tcaactgttg tagcaaacag tgacgaatct cagcttctga caccaggaaa gatgagtcag
     1501 cgccaaggaa aagaagctta tccaacgcca accaaagatt tgcatcagcc atctcttagt
     1561 ccagcaagtc ctcatagcca gggttttgaa agagggaagg aagatatttc tcaaaataaa
     1621 gatgaatctt cactttctat gtcaaagagc aagtctgaat ctaaacttta taatggctca
     1681 gagaaggaca gttcaacttc aagcaaactc acaaaaaaag aatctcttaa ggtacaaaag
     1741 aaaaattacc gagaagaaaa gaaaagagcc acaaaggagc tgctcagtac aatcacagat
     1801 ccttctgtta ttgttatggc tgattggtta aagattcgtg gtactctaaa gagctggacc
     1861 aagttatggt gtgtgttgaa acctggggtg ctactgatct ataaaaccca aaaaaatggt
     1921 cagtgggtag gaacagttct tctgaatgcc tgtgaaatca ttgaacgtcc atcaaaaaag
     1981 gatggctttt gtttcaaact tttccatcct ttggagcaat ctatttgggc agtgaagggt
     2041 ccaaaaggtg aagcggttgg atccattact caacccttac ctagcagtta tttgatcatc
     2101 cgagctactt cagagtcaga tggaaggtgc tggatggatg ctttggagtt ggctttgaaa
     2161 tgttctagtc ttcttaaacg tacaatgatc agagaaggaa aggaacatga cctgagcgtt
     2221 tcatcagata gcacacatgt gactttctat ggcttactac gtgctaacaa tctccacagt
     2281 ggtgataact tccagttaaa tgatagtgaa attgaacgac aacattttaa ggaccaagat
     2341 atgtattctg ataaatctga taaagaaaat gatcaagaac atgatgagtc tgataatgag
     2401 gtgatgggga aaagtgaaga aagtgacaca gatacatcag aaagacaaga tgactcatat
     2461 atcgaacctg agcctgttga gcctttaaag gagactacct acactgaaca gagccatgaa
     2521 gaacttggag aggcaggtga ggcttctcaa acagaaactg tatctgaaga aaacaaaagc
     2581 cttatctgga cactattgaa acaagtccgt cctggcatgg acctatccaa ggtggttctg
     2641 cctacattta ttttggaacc ccgttctttc ctggataaac tttcagatta ctactatcat
     2701 gcagatttcc tatctgaggc agctcttgaa gaaaatcctt atttccgttt gaaaaaggtc
     2761 gtaaagtggt atttgtcagg attctataaa aagccaaagg gactgaagaa accttataat
     2821 cctatacttg gcgagacttt ccgttgttta tggattcatc ccagaacaaa cagcaaaact
     2881 ttttatattg ctgaacaggt gtcccatcat ccaccaatat ctgcctttta tgttagtaat
     2941 cgaaaagatg gattttgcct tagcggtagt atcctggcta agtctaagtt ttatggaaac
     3001 tcattatctg caatattaga gggagaagca cggttaactt tcttgaatag aggtgaagat
     3061 tatgtaatga caatgccata cgctcattgt aaaggaattc tttatggtac aatgacactg
     3121 gagcttggtg gaacagtcaa tattacatgt caaaaaactg gatacagtgc aatacttgaa
     3181 tttaaactaa agccattcct agggagtagt gactgtgtta atcaaatatc agggaaactt
     3241 aaactgggaa aagaagtcct agctactttg gaaggtcatt gggatagtga agtttttatt
     3301 actgataaaa agactgataa ttcagaggtt ttctggaatc caacacctga cattaagcaa
     3361 tggagattaa taaggcacac tgtaaaattt gaagaacagg gagattttga atcagagaaa
     3421 ctctggcaac gggtaactcg agccataaat gccaaagacc aaactgaagc tacccaagag
     3481 aagtatgttt tggaagaagc tcaaagacaa gctgccaggg atcggaaaac aaaaaatgaa
     3541 gagtggtctt gcaaattatt tgaacttgat ccactcacag gagaatggca ttacaagttt
     3601 gcagataccc gaccatggga cccacttaat gatatgatac agtttgaaaa agatggtgtt
     3661 attcagacca aagtgaaaca tcgtactcca atggttagcg tccccaaaat gaaacataag
     3721 ccaaccaggc aacagaagaa agtagcaaaa ggctattcct ccccagaacc tgacattcaa
     3781 gactcctctg gaagtgaagc tcaatcagta aaaccaagta caagaagaaa gaaaggaata
     3841 gaactgggag acattcagag ttccatcgaa tctataaaac aaacacagga agaaattaaa
     3901 agaaatatta tggctcttcg aaatcattta gtttcaagca caccggccac ggattatttt
     3961 ctgcaacaaa aagactactt catcattttc ctcctgattt tgcttcaagt cataataaac
     4021 ttcatgttca agtagaccgc gggcccggga tccaccggat ctagataact gatcataatc
     4081 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     4141 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     4201 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4261 tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa
     4321 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     4381 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     4441 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     4501 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     4561 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4621 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4681 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4741 tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4801 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4861 aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat
     4921 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4981 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     5041 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5101 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5161 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     5221 ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt
     5281 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     5341 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     5401 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     5461 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     5521 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     5581 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     5641 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     5701 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     5761 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc
     5821 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5881 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     5941 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     6001 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     6061 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     6121 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     6181 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     6241 ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     6301 cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat
     6361 aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg
     6421 ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc
     6481 ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat
     6541 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6601 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6661 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6721 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6781 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6841 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6901 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6961 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7021 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7081 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7141 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7201 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7261 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7321 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7381 gccatgcat
//
