LOCUS       sequence-472413-        8443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCUB with MCU N-terminal domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8443)
  AUTHORS   Rai NK, Eberhardt DR, Balynas AM, MacEwen MJS, Bratt AR, Sancak Y, 
            Chaudhuri D
  TITLE     Mechanism of MCUB-Dependent Inhibition of Mitochondrial Calcium 
            Uptake.
  JOURNAL   J Cell Physiol. 2025 Apr;240(4):e70033. doi: 10.1002/jcp.70033.
  PUBMED    40227803
REFERENCE   2  (bases 1 to 8443)
  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: "J Cell 
            Physiol."; date: "2025-04"; volume: "240(4):e70033. doi"; pages: " 
            10.1002/jcp.70033"
COMMENT     Sequence Label: MCUb-MCU-NTD-pLYS1-Flag
FEATURES             Location/Qualifiers
     source          1..8443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        131..334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     285..305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1452..1475
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1526..1643
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1692..2202
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2121..2139
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2224..2823
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2224..2243)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2720..2740
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2951..3184
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3256..3377
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3293..3312)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3347..3366
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3417..3552
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3572..3591)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3573..3591)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3601..3618)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3601..3617)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3610..3632)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3759..4214
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3846..3865)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4056..4077
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4240..4344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4345..4413,4414..5205)
                     /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(4563..4582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5376..5964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5865..5884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6118..6135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6252..6273
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6288..6293,6294..6311,6312..6318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6326..6342
                     /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     6331..6353
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6350..6366
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6350..6366
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6385..6405
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6387..6405
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6433..6659
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6660..6840
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6887..7012
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7505..7738
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7923..7967
                     /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"
     primer_bind     8265..8285
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
       61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      361 gcatggcggc cgccgcaggt agatcgctcc tgctgctcct ctcctctcgg ggcggcggcg
      421 gcgggggcgc cggcggctgc ggggcgctga ctgccggctg cttccctggg ctgggcgtca
      481 gccgccaccg gcagcagcag caccaccgga cggtacacca gaggatcgct tcctggcaga
      541 atttgggagc tgtttattgc agcactgttg tgccctctga tgatgttaca gtggtttatc
      601 aaaatgggtt acctgtgata tctgtgaggc taccatcccg gcgtgaacgc tgtcagttca
      661 cactcaagcc tatctctgac tctgttggtg tatttttacg acaactgcaa gaagaggatc
      721 ggggaattga cagagttgct atctattcac cagatggtgt tcgcgttgct gcttcaacag
      781 gaatagacct cctcctcctt gatgacttta agctggtcat taatgactta acataccacg
      841 tacgaccacc aaaaagagac ctcttaagtc atgaaaatgc agcaacgctg aatgatgtaa
      901 agacattggt ccagcaacta tttacaatct tgcatttaga agagtctcag aaaaagagag
      961 agcaccattt actggagaaa attgaccacc tgaaggaaca gctgcagccc cttgaacagg
     1021 tgaaagctgg aatagaagct cattcggaag ccaaaaccag tggactcctg tgggctggat
     1081 tggcactgct gtccattcag ggtggggcac tggcctggct cacgtggtgg gtgtactcct
     1141 gggatatcat ggagccagtt acatacttca tcacatttgc aaattctatg gtcttttttg
     1201 catactttat agtcactcga caggattata cttactcagc tgttaagagt aggcaatttc
     1261 ttcagttctt ccacaagaaa tcaaagcaac agcactttga tgtgcagcaa tacaacaagt
     1321 taaaagaaga ccttgctaag gctaaagaat ccctgaaaca ggcgcgtcat tctctctgtt
     1381 tgcaaatgca agtagaagaa ctcaatgaaa agaattctag aggtggatct ggtggatctg
     1441 gtggatctat ggattacaag gatgacgatg acaagtaagg taccgaattc tcgacctcga
     1501 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     1561 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     1621 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc
     1681 cgcggctcga gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga
     1741 cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca
     1801 ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg
     1861 gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg
     1921 acgtgacaaa cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga
     1981 gcaatggcag cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc
     2041 gccgagagca gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg
     2101 gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca
     2161 gtcggctccc tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt
     2221 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     2281 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     2341 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     2401 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     2461 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     2521 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     2581 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     2641 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     2701 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     2761 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     2821 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg
     2881 gtacctttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa
     2941 aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt
     3001 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     3061 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
     3121 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     3181 agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata
     3241 tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3301 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3361 ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc
     3421 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3481 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3541 tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc
     3601 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     3661 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     3721 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     3781 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3841 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3901 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3961 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4021 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4081 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     4141 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4201 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4261 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4321 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4381 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4441 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4501 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4561 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4621 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4681 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4741 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4801 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4861 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4921 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4981 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5041 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5101 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5161 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5221 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5281 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5341 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5401 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5461 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5521 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5581 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5641 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5701 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5761 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5821 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5881 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5941 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6001 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     6061 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     6121 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     6181 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6241 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     6301 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6361 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     6421 ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga
     6481 gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa
     6541 ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa
     6601 ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg
     6661 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     6721 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     6781 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     6841 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
     6901 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
     6961 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
     7021 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
     7081 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
     7141 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
     7201 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
     7261 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
     7321 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
     7381 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     7441 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     7501 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     7561 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     7621 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     7681 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     7741 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     7801 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     7861 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     7921 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     7981 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     8041 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     8101 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     8161 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac
     8221 ggtatcgatc acgagactag cctcgagcgg ccgccccctt caccgagggc ctatttccca
     8281 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     8341 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     8401 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tca
//
