LOCUS       sequence-173225-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of ATP8 with G2 mitochondrial targeting 
            sequence and V5 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  AUTHORS   Boominathan A, Vanhoozer S, Basisty N, Powers K, Crampton AL, Wang 
            X, Friedricks N, Schilling B, Brand MD, O'Connor MS
  TITLE     Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA 
            Complex V null mutant.
  JOURNAL   Nucleic Acids Res. 2016 Nov 2;44(19):9342-9357. Epub 2016 Sep 4.
  PUBMED    27596602
REFERENCE   2  (bases 1 to 6508)
  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: "Nucleic 
            Acids Res."; date: "2016-11-2"; volume: "44(19)"; pages: "9342-9357.
            Epub 2016 Sep 4"
COMMENT     Sequence Label: pCMV6-G2-ATP8-V5 (puromycin)
FEATURES             Location/Qualifiers
     source          1..6508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      10..465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(97..116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     307..328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     591..613
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     605..622
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     606..622
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        783..1162
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1163..1366
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1317..1337
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1392..1411
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1392..1410
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2069..2110
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2295..2311)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2352..2368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2379..3001
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2578..2597)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3025..3045)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3030..3359
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3210..3345
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3417..4016
                     /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(3417..3436)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3913..3933
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4195..4316
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4232..4251)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4286..4305
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4365..4381)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4378..4400)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4389..4405
                     /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)."
     promoter        complement(join(4413..4419,4420..4437,4438..4443))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4458..4479
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4596..4613)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4767..5355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4847..4866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5526..6317,6318..6386))
                     /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     6149..6168
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6387..6491)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
       61 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      121 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      181 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      241 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
      301 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
      361 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
      421 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttccatt cgccattcag
      481 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
      541 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
      601 acgttgtaaa acgacggcca gtgccaagct gatctataca ttgaatcaat attggcaatt
      661 agccatatta gtcattggtt atatagcata aatcaatatt ggctattggc cattgcatac
      721 gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat gaccgccatg
      781 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      841 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      901 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      961 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     1021 tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc
     1081 ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt
     1141 attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata
     1201 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     1261 ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca
     1321 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     1381 tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt
     1441 accgaggaga tctgccgccg cgatcgccat gcctgagctg atcctgtatg ttgcaatcac
     1501 tctatccgtg gctgagcgac tcgttggccc gggtcacgca tgcgctgagc cttcctttcg
     1561 ctcttcccgc tgctccgccc ctctctgtct tctctgcagt gggagcagct ctcctgccac
     1621 agctcctcac cccctgaaaa tgttcgcctg ctccaagttt gtctccactc cctccttggt
     1681 gaagagcacc tcacagctgc tgagccgtcc gctatctgca gtggtgctga aacgaccgga
     1741 gatactgaca gatgagagcc tcagcagctt ggcagtctca tgtcccctta cctcacttgt
     1801 ctctagccgc agcttccaaa ccagcgccat ttcaagggac atcgacacag caacgcgtat
     1861 gccccagctg aacaccacag tgtggcctac tatgatcacc ccaatgctgc tgacactgtt
     1921 cctgattact cagctgaaga tgctgaacac caattaccac ctgccccctt ccccaaaacc
     1981 catgaagatg aaaaactata ataagccctg ggaacctaag tggaccaaaa tctgtagcct
     2041 gcatagcctg ccccctcagt cactcgaggg taagcctatc cctaaccctc tcctcggtct
     2101 cgattctacg taggcggccg cggccggcca ggagccgtct ccacctccca tagttctccc
     2161 gcgtctggtt ggccccgtgt gttccttttc ctatacctcc ccaggcagcc tggggaacgt
     2221 ggttggctca gggtttgaca gagaaaagac aaataaatac tgtattaata agatgtttct
     2281 tgaggccggc cgcggtcata gctgtttcct gaacagatcc tctagagata tcgtttaaac
     2341 ggccggccgc ggtcatagct gtttcctgaa cagatcccgg gtggcatccc tgtgacccct
     2401 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     2461 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     2521 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     2581 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     2641 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     2701 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     2761 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     2821 tacaggcgtg aaccactgct cccttccctg tccttctgat tttaaaataa ctataccagc
     2881 aggaggacgt ccagacacag cataggctac ctggccatgc ccaaccggtg ggacatttga
     2941 gttgcttgct tggcactgtc ctctcatgcg ttgggtccac tcagtagatg cctgttgaat
     3001 tgggtacgcg gccagcttgg ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3061 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3121 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3181 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3241 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3301 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3361 agctcccggg agcttgtata tccattttcg gatctgatca agagacacgt acgaccatga
     3421 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtcccccgg gcagtacgca
     3481 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccagaccgcc
     3541 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3601 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3661 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3721 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3781 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3841 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3901 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3961 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgactgc
     4021 agtgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4081 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4141 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4201 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4261 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4321 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4381 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4441 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4501 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4561 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4621 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4681 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4741 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4801 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4861 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4921 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4981 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5041 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5101 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5161 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5221 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5281 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5341 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5401 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5461 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5521 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5581 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5641 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5701 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5761 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5821 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5881 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5941 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6001 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6061 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6121 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6181 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6241 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6301 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6361 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6421 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6481 ggggttccgc gcacatttcc ccgaaaag
//
