LOCUS       sequence-75127-e        7651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human EMRE in mammalian cells with silent mutation 
            making it resistant to RNAi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7651)
  AUTHORS   Sancak Y, Markhard AL, Kitami T, Kovacs-Bogdan E, Kamer KJ, Udeshi 
            ND, Carr SA, Chaudhuri D, Clapham DE, Li AA, Calvo SE, Goldberger O,
            Mootha VK
  TITLE     EMRE is an Essential Component of the Mitochondrial Calcium 
            Uniporter Complex.
  JOURNAL   Science. 2013 Nov 14.
  PUBMED    24231807
REFERENCE   2  (bases 1 to 7651)
  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: "Science. 
            2013 Nov 14."
COMMENT     Sequence Label: pLYS1-EMRE/RNAi resistant
FEATURES             Location/Qualifiers
     source          1..7651
                     /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"
     misc_feature    734..851
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        900..1410
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1329..1347
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1432..2031
                     /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(1432..1451)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1928..1948
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2159..2392
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2464..2585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2501..2520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2555..2574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2625..2760
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2780..2799)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2781..2799)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2809..2826)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2809..2825)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2818..2840)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2967..3422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3054..3073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3264..3285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3448..3552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3553..3621,3622..4413)
                     /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(3771..3790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4584..5172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5073..5092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5326..5343
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5460..5481
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5496..5501,5502..5519,5520..5526)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5534..5550
                     /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     5539..5561
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5558..5574
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5558..5574
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5593..5613
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5595..5613
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5641..5867
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5868..6048
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6095..6220
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6713..6946
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7131..7175
                     /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     7473..7493
                     /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 gcatggcgtc cggagcggct cgctggctag tattggcacc cgtcaggtcc ggggctctcc
      421 ggagcgggcc tagcttgagg aaagatggcg atgtctccgc cgcatggagc ggctcaggcc
      481 ggagcctagt tccatcgagg tcagtcatcg ttacccgcag cggcgccatt ttgcccaagc
      541 cagtcaaaat gtccttcggc cttctccgtg tgttctccat tgtgatcccc tttctctatg
      601 tcgggacact cattagcaaa aatttcgcgg cactattaga ggaacatgac atttttgttc
      661 cagaggatga tgatgatgat gactaaggta ccgaattctc gacctcgaga caaatggcag
      721 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
      781 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
      841 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
      901 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
      961 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     1021 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     1081 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     1141 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     1201 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     1261 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     1321 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     1381 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     1441 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     1501 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     1561 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     1621 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     1681 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     1741 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     1801 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     1861 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     1921 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     1981 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     2041 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     2101 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     2161 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     2221 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     2281 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     2341 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     2401 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     2461 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2521 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2581 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     2641 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2701 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2761 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     2821 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     2881 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     2941 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3001 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3061 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3121 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3181 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3241 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3301 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3361 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3421 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3481 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3541 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3601 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3661 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3721 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3781 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3841 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3901 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     3961 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4021 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4081 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4141 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4201 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4261 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4321 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4381 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4441 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4501 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4561 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4621 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4681 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4741 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4801 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4861 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4921 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4981 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5041 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5101 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5161 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5221 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5281 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5341 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5401 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5461 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5521 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5581 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa
     5641 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
     5701 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
     5761 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
     5821 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
     5881 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     5941 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     6001 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
     6061 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
     6121 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
     6181 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
     6241 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
     6301 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
     6361 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
     6421 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
     6481 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
     6541 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
     6601 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     6661 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     6721 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     6781 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     6841 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     6901 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     6961 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     7021 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     7081 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     7141 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     7201 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     7261 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     7321 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     7381 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
     7441 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
     7501 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     7561 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     7621 ttttaaaatt atgttttaaa atggactatc a
//
