LOCUS       sequence-429910-        7251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of fluorescent N-terminally tagged H514A-H515A 
            mutant of ORP8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7251)
  AUTHORS   Criado Santos N, Bouvet S, Cruz Cobo M, Mandavit M, Bermont F, 
            Castelbou C, Mansour F, Azam M, Giordano F, Nunes-Hasler P
  TITLE     Sec22b regulates phagosome maturation by promoting ORP8-mediated 
            lipid exchange at endoplasmic reticulum-phagosome contact sites.
  JOURNAL   Commun Biol. 2023 Oct 4;6(1):1008. doi: 10.1038/s42003-023-05382-0.
  PUBMED    37794132
REFERENCE   2  (bases 1 to 7251)
  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: "Commun 
            Biol."; date: "2023-10-4"; volume: "6(1):1008. doi"; pages: " 
            10.1038/s42003-023-05382-0"
COMMENT     Sequence Label: pEGFP-ORP8-H514A-H515A
FEATURES             Location/Qualifiers
     source          1..7251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1066..1187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1103..1122)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1157..1176
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1194..1649)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1331..1352)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1543..1562
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1676..1780
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1782..2139
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1782..1973
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1805..1825)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1990..2125
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2174..2968
                     /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             2174..2968
                     /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(2228..2247)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2838..2857
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3156..3175)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3200..3247
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3576..4164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4065..4084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4339..4642
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4643..4846
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4797..4817
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4885..4894
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4891..4893,4894..4896,4897..5607)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4891..4893,4894..4896,4897..5607)
                     /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(4936..4957)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5197..5216)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5544..5565
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gagcttggtg gaacagtcaa tattacatgt caaaaaactg gatacagtgc aatacttgaa
       61 tttaaactaa agccattcct agggagtagt gactgtgtta atcaaatatc agggaaactt
      121 aaactgggaa aagaagtcct agctactttg gaaggtcatt gggatagtga agtttttatt
      181 actgataaaa agactgataa ttcagaggtt ttctggaatc caacacctga cattaagcaa
      241 tggagattaa taaggcacac tgtaaaattt gaagaacagg gagattttga atcagagaaa
      301 ctctggcaac gggtaactcg agccataaat gccaaagacc aaactgaagc tacccaagag
      361 aagtatgttt tggaagaagc tcaaagacaa gctgccaggg atcggaaaac aaaaaatgaa
      421 gagtggtctt gcaaattatt tgaacttgat ccactcacag gagaatggca ttacaagttt
      481 gcagataccc gaccatggga cccacttaat gatatgatac agtttgaaaa agatggtgtt
      541 attcagacca aagtgaaaca tcgtactcca atggttagcg tccccaaaat gaaacataag
      601 ccaaccaggc aacagaagaa agtagcaaaa ggctattcct ccccagaacc tgacattcaa
      661 gactcctctg gaagtgaagc tcaatcagta aaaccaagta caagaagaaa gaaaggaata
      721 gaactgggag acattcagag ttccatcgaa tctataaaac aaacacagga agaaattaaa
      781 agaaatatta tggctcttcg aaatcattta gtttcaagca caccggccac ggattatttt
      841 ctgcaacaaa aagactactt catcattttc ctcctgattt tgcttcaagt cataataaac
      901 ttcatgttca agtaggtcga cggtaccgcg ggcccgggat ccaccggatc tagataactg
      961 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     1021 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     1081 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     1141 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     1201 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     1261 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     1321 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     1381 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     1441 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     1501 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     1561 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     1621 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     1681 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1741 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     1801 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     1861 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     1921 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     1981 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     2041 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     2101 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     2161 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     2221 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     2281 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     2341 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     2401 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     2461 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     2521 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     2581 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     2641 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     2701 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     2761 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     2821 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     2881 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     2941 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     3001 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     3061 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     3121 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     3181 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     3241 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     3301 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     3361 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     3421 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     3481 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     3541 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3601 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3661 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3721 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3781 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3841 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3901 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3961 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4021 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4081 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4141 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4201 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4261 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
     4321 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     4381 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     4441 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     4501 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     4561 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     4621 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     4681 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     4741 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     4801 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     4861 tcagatccgc tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
     4921 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     4981 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     5041 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     5101 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     5161 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     5221 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     5281 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     5341 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     5401 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     5461 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     5521 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     5581 actctcggca tggacgagct gtacaagtcc ggactcagat ctatgagtca gcgccaagga
     5641 aaagaagctt atccaacgcc aaccaaagat ttgcatcagc catctcttag tccagcaagt
     5701 cctcatagcc agggttttga aagagggaag gaagatattt ctcaaaataa agatgaatct
     5761 tcactttcta tgtcaaagag caagtctgaa tctaaacttt ataatggctc agagaaggac
     5821 agttcaactt caagcaaact cacaaaaaaa gaatctctta aggtacaaaa gaaaaattac
     5881 cgagaagaaa agaaaagagc cacaaaggag ctgctcagta caatcacaga tccttctgtt
     5941 attgttatgg ctgattggtt aaagattcgt ggtactctaa agagctggac caagttatgg
     6001 tgtgtgttga aacctggggt gctactgatc tataaaaccc aaaaaaatgg tcagtgggta
     6061 ggaacagttc ttctgaatgc ctgtgaaatc attgaacgtc catcaaaaaa ggatggcttt
     6121 tgtttcaaac ttttccatcc tttggagcaa tctatttggg cagtgaaggg tccaaaaggt
     6181 gaagcggttg gatccattac tcaaccctta cctagcagtt atttgatcat ccgagctact
     6241 tcagagtcag atggaaggtg ctggatggat gctttggagt tggctttgaa atgttctagt
     6301 cttcttaaac gtacaatgat cagagaagga aaggaacatg acctgagcgt ttcatcagat
     6361 agcacacatg tgactttcta tggcttacta cgtgctaaca atctccacag tggtgataac
     6421 ttccagttaa atgatagtga aattgaacga caacatttta aggaccaaga tatgtattct
     6481 gataaatctg ataaagaaaa tgatcaagaa catgatgagt ctgataatga ggtgatgggg
     6541 aaaagtgaag aaagtgacac agatacatca gaaagacaag atgactcata tatcgaacct
     6601 gagcctgttg agcctttaaa ggagactacc tacactgaac agagccatga agaacttgga
     6661 gaggcaggtg aggcttctca aacagaaact gtatctgaag aaaacaaaag ccttatctgg
     6721 acactattga aacaagtccg tcctggcatg gacctatcca aggtggttct gcctacattt
     6781 attttggaac cccgttcttt cctggataaa ctttcagatt actactatca tgcagatttc
     6841 ctatctgagg cagctcttga agaaaatcct tatttccgtt tgaagaaagt agtgaaatgg
     6901 tatttgtcag gattctataa aaagccaaag ggactgaaga aaccttataa tcctatactt
     6961 ggcgagactt tccgttgttt atggattcat cccagaacaa acagcaaaac tttttatatt
     7021 gctgaacagg tgtccgctgc tccaccaata tctgcctttt atgttagtaa tcgaaaagat
     7081 ggattttgcc ttagcggtag tatcctggct aagtctaagt tttatggaaa ctcattatct
     7141 gcaatattag agggagaagc acggttaact ttcttgaata gaggtgaaga ttatgtaatg
     7201 acaatgccat acgctcattg taaaggaatt ctttatggta caatgacact g
//
