LOCUS       sequence-458833-        8028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a synthetic linker, tagged with mRFP, stabilizing the 
            ER-mitochondrial distance at 20 nm from a lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8028)
  AUTHORS   Dematteis G, Tapella L, Casali C, Talmon M, Tonelli E, Reano S, 
            Ariotti A, Pessolano E, Malecka J, Chrostek G, Kulkoviene G, 
            Umbrasas D, Distasi C, Grilli M, Ladds G, Filigheddu N, Fresu LG, 
            Mikoshiba K, Matute C, Ramos-Gonzalez P, Jekabsone A, Cali T, Brini 
            M, Biggiogera M, Cavaliere F, Miggiano R, Genazzani AA, Lim D
  TITLE     ER-mitochondria distance is a critical parameter for efficient 
            mitochondrial Ca(2+) uptake and oxidative metabolism.
  JOURNAL   Commun Biol. 2024 Oct 10;7(1):1294. doi: 10.1038/s42003-024-06933-9.
  PUBMED    39390051
REFERENCE   2  (bases 1 to 8028)
  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: "2024-10-10"; volume: "7(1):1294. doi"; pages: " 
            10.1038/s42003-024-06933-9"
COMMENT     Sequence Label: pLV-20nmEML-mRFP
FEATURES             Location/Qualifiers
     source          1..8028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        62..365
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        366..569
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     520..540
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     566..590
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             991..1662
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     misc_feature    1993..2581
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2046..2066)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2464..2475)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2668..2901
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2973..3094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3010..3029)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3064..3083
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3134..3269
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3289..3308)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3290..3308)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3318..3335)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3318..3334)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3327..3349)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3476..3931
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3563..3582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3773..3794
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3957..4061
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4062..4130,4131..4922)
                     /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     complement(4280..4299)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5093..5681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5582..5601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5835..5852
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5969..5990
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6005..6010,6011..6028,6029..6035)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6043..6059
                     /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)."
     primer_bind     6048..6070
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6067..6083
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6067..6083
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6102..6122
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6104..6122
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6150..6376
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6377..6557
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6604..6729
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7222..7455
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7640..7684
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    join(7951..8028,1..39)
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 taaagaatta caaaaacaaa ttacaaaatt caaaatttta tcgataagct tgggagttcc
       61 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      121 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      181 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      241 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      301 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      361 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      421 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      481 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      541 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
      601 gccatccacg ctgttttgac ctccatagaa gacaccgact ctagcatggc gattcaactg
      661 cgtagcctgt ttccgctggc gctgccgggt ctgctggcgc tgctgggttg gtggtggttc
      721 tttagccgta agaaggaccc gacccgtagc gcgaacaaac agcaagagga agaggcggag
      781 cgtctgcgtc gtatccagga agagatggag aaggaacgta aacgtcgtga agaggatgaa
      841 caacgtcgtc gtaaggaaga ggaagagcgt cgtatgaaac tggagatgga agcgaagcgt
      901 aaacaggaag aggaagagcg taagaaacgt gaagacgatg agaagcgtat ccaagcggag
      961 cgtaacagcg acccgccggt ggcgaccctg gcgagcagcg aggatgttat caaggaattc
     1021 atgcgtttca aagtgcgtat ggagggcagc gttaacggtc acgagttcga aattgagggt
     1081 gaaggcgagg gtcgtccgta cgaaggtacc cagaccgcga agctgaaagt gaccaaaggt
     1141 ggcccgctgc cgtttgcgtg ggacatcctg agcccgcagt ttcaatacgg cagcaaggcg
     1201 tatgttaaac acccggcgga cattccggat tatctgaagc tgagcttccc ggagggtttt
     1261 aaatgggaac gtgtgatgaa ctttgaggat ggtggcgtgg ttaccgttac ccaggacagc
     1321 agcctgcaag atggcgaatt catctacaag gtgaaactgc gtggcaccaa ctttccgagc
     1381 gacggtccgg ttatgcagaa gaaaaccatg ggttgggagg cgagcaccga acgtatgtat
     1441 ccggaggatg gcgcgctgaa gggtgaaatt aaaatgcgtc tgaagctgaa agacggtggc
     1501 cactacgatg cggaggtgaa gaccacctat atggcgaaga aaccggttca actgccgggc
     1561 gcgtacaaga ccgacatcaa actggatatt accagccaca acgaagacta caccatcgtg
     1621 gaacagtatg agcgtgcgga aggtcgtcac agcaccggtg cgagcggtct gcgtagccgt
     1681 gcgcaagcga gcaacagcaa acagcaagaa gaggaagcgg agcgtctgcg ccgcattcag
     1741 gaggaaatgg aaaaggagcg taaacgtcgt gaggaagacg agcaacgtcg tcgtaaggag
     1801 gaagaggaac gtcgtatgaa actggaaatg gaggcgaagc gtaaacagga ggaagaggag
     1861 cgtaagaaac gtgaggacga tgaaaagcgt atccaggcgg agagccgtgc gcaggcgagc
     1921 aacagccgtg ttatggtgta tattggtatt gcgatttttc tgtttgttgg tctgtttatg
     1981 aagtaactcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     2041 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     2101 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     2161 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     2221 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     2281 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     2341 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt
     2401 ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     2461 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     2521 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     2581 cctggaattc gagctcggta cctttaagac caatgactta caaggcagct gtagatctta
     2641 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     2701 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     2761 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     2821 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     2881 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
     2941 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     3001 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     3061 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     3121 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3181 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3241 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     3301 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     3361 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     3421 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     3481 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3541 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3601 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3661 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3721 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3781 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3841 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3901 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     3961 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4021 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4081 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4141 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4201 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4261 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4321 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4381 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4441 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4501 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4561 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4621 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4681 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4741 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4801 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4861 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4921 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4981 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5041 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5101 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5161 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5221 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5281 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5341 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5401 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5461 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5521 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5581 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5641 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5701 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5761 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5821 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     5881 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     5941 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     6001 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     6061 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     6121 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
     6181 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
     6241 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
     6301 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
     6361 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     6421 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     6481 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     6541 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
     6601 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
     6661 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     6721 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     6781 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     6841 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     6901 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     6961 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     7021 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     7081 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     7141 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     7201 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     7261 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     7321 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     7381 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     7441 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     7501 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     7561 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     7621 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     7681 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     7741 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     7801 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     7861 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     7921 agtgaacgga tctcgacggt atcggttaac ttttaaaaga aaagggggga ttggggggta
     7981 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaac
//
