LOCUS       sequence-458832-        8232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a synthetic linker, tagged with mRFP, stabilizing the 
            ER-mitochondrial distance at 30 nm from a lentiviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8232)
  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 8232)
  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-30nmEML-mRFP
FEATURES             Location/Qualifiers
     source          1..8232
                     /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             1195..1866
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(1322..1340)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    2197..2785
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2250..2270)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2668..2679)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2872..3105
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3177..3298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3214..3233)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3268..3287
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3338..3473
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3493..3512)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3494..3512)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3522..3539)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3522..3538)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3531..3553)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3680..4135
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3767..3786)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3977..3998
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4161..4265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4266..4334,4335..5126)
                     /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(4484..4503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5297..5885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5786..5805
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6039..6056
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6173..6194
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6209..6214,6215..6232,6233..6239)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6247..6263
                     /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     6252..6274
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6271..6287
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6271..6287
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6306..6326
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6308..6326
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6354..6580
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6581..6761
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6808..6933
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7426..7659
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7844..7888
                     /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(8155..8232,1..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 tattcagctg
      661 cgctcactgt ttcctctggc tctgcctgga ctgctggccc tgctggggtg gtggtggttc
      721 ttttcccgca aaaaggaccc cacccgctcc gccaacaagc agcaggaaga ggaggcagag
      781 cggctgcgga gaatccagga ggagatggag aaggagagaa agaggcgcga ggaggatgag
      841 cagcggagaa ggaaagagga ggaggagcgc cggatgaagc tggagatgga ggccaagcgg
      901 aagcaggagg aggaggagag aaagaagagg gaggacgatg agaagagaat ccaggccgag
      961 aaacagcagg aagaggaggc cgagaggctg agaaggattc aggaagagat ggagaaggaa
     1021 aggaagagga gggaggagga cgagcagaga aggcgcaagg aggaggagga gcggagaatg
     1081 aaactggaaa tggaagctaa aagaaaacag gaagaggagg agcgcaagaa gcgggaagat
     1141 gatgagaaga ggatccaggc agagaggaac agcgacccac ctgtggccac actggccagc
     1201 tccgaggatg tgatcaagga gttcatgcgc tttaaagtgc ggatggaggg ctccgtgaat
     1261 ggccacgagt tcgagatcga gggagaggga gagggcagac cctacgaggg cacccagaca
     1321 gccaagctga aggtgaccaa gggaggacca ctgcccttcg cctgggacat cctgtctcct
     1381 cagtttcagt acggcagcaa ggcctatgtg aagcaccctg ccgacatccc agattatctg
     1441 aagctgtctt tcccagaggg ctttaagtgg gagagggtga tgaactttga ggatggcggc
     1501 gtggtgaccg tgacacagga ctctagcctg caggatggcg agttcatcta caaggtgaag
     1561 ctgcgcggca caaattttcc ctccgacggc cctgtgatgc agaagaagac catgggctgg
     1621 gaggcctcta cagagaggat gtatcctgag gatggcgccc tgaagggcga gatcaagatg
     1681 cgcctgaagc tgaaggacgg cggccactac gatgccgagg tgaagaccac atatatggcc
     1741 aagaagccag tgcagctgcc aggagcctac aagaccgaca tcaagctgga tatcacaagc
     1801 cacaacgagg actacaccat cgtggagcag tatgagagag ccgagggcag gcacagcacc
     1861 ggagcctccg gcctgcgctc tcgggcacag gcctccaatt ctaaacagca ggaagaagag
     1921 gcagagcggc tgaggcgcat tcaggaagaa atggaaaaag agagaaagcg gagagaggag
     1981 gatgagcaga ggcgccggaa agaagaggag gagagaagaa tgaaactgga gatggaagct
     2041 aaaagaaagc aggaagaaga agaaagaaag aagagggaag atgacgaaaa gagaatccag
     2101 gcagagagca gggcacaggc ctccaattca agggtgatgg tctacattgg gattgctatt
     2161 tttctgtttg tcggactgtt tatgaagtga ctcgacaatc aacctctgga ttacaaaatt
     2221 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     2281 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     2341 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     2401 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     2461 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     2521 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     2581 ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg
     2641 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     2701 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     2761 atctcccttt gggccgcctc cccgcctgga attcgagctc ggtaccttta agaccaatga
     2821 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     2881 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     2941 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     3001 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     3061 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     3121 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact
     3181 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3241 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3301 atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3361 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3421 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac
     3481 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     3541 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     3601 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     3661 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3721 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3781 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3841 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3901 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3961 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4021 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4081 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     4141 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4201 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4261 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4321 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4381 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4441 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4501 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4561 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4621 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4681 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4741 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4801 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4861 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4921 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4981 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5041 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5101 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5161 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5221 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5281 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5341 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5401 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5461 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5521 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5581 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5641 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5701 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5761 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5821 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5881 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5941 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     6001 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6061 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     6121 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     6181 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     6241 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     6301 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
     6361 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
     6421 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
     6481 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
     6541 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
     6601 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     6661 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     6721 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     6781 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
     6841 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
     6901 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
     6961 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
     7021 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     7081 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     7141 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     7201 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     7261 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     7321 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     7381 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     7441 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     7501 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     7561 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     7621 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     7681 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     7741 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     7801 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     7861 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     7921 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     7981 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     8041 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     8101 agacagagac agatccattc gattagtgaa cggatctcga cggtatcggt taacttttaa
     8161 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     8221 agacatacaa ac
//
