LOCUS       sequence-170395-        6304 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 6304)
  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 6304)
  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..6304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     151..173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     165..182
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     166..182
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        723..926
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     952..971
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        952..970
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1629..1670
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1708..1724)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1735..2357
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1934..1953)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2381..2401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2386..2715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2566..2701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2773..3372
                     /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(2773..2792)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3269..3289
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3551..3672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3588..3607)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3642..3661
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3721..3737)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3734..3756)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3745..3761
                     /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(3769..3775,3776..3793,3794..3799))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3814..3835
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3952..3969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4123..4711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4203..4222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4882..5673,5674..5742))
                     /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     5505..5524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5743..5847)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5874..6302
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5961..5980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6171..6192
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac tgttgggaag
       61 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      121 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      181 gtgccaagct gatctataca ttgaatcaat attggcaatt agccatatta gtcattggtt
      241 atatagcata aatcaatatt ggctattggc cattgcatac gttgtatcta tatcataata
      301 tgtacattta tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagaatttt gtaatacgac
      961 tcactatagg gcggccggga attcgtcgac tggatccggt accgaggaga tctgccgccg
     1021 cgatcgccat gcctgagctg atcctgtatg ttgcaatcac tctatccgtg gctgagcgac
     1081 tcgttggccc gggtcacgca tgcgctgagc cttcctttcg ctcttcccgc tgctccgccc
     1141 ctctctgtct tctctgcagt gggagcagct ctcctgccac agctcctcac cccctgaaaa
     1201 tgttcgcctg ctccaagttt gtctccactc cctccttggt gaagagcacc tcacagctgc
     1261 tgagccgtcc gctatctgca gtggtgctga aacgaccgga gatactgaca gatgagagcc
     1321 tcagcagctt ggcagtctca tgtcccctta cctcacttgt ctctagccgc agcttccaaa
     1381 ccagcgccat ttcaagggac atcgacacag caacgcgtat gccccagctg aacaccacag
     1441 tgtggcctac tatgatcacc ccaatgctgc tgacactgtt cctgattact cagctgaaga
     1501 tgctgaacac caattaccac ctgccccctt ccccaaaacc catgaagatg aaaaactata
     1561 ataagccctg ggaacctaag tggaccaaaa tctgtagcct gcatagcctg ccccctcagt
     1621 cactcgaggg taagcctatc cctaaccctc tcctcggtct cgattctacg taggcggccg
     1681 cactcgaggt ttaaacggcc ggccgcggtc atagctgttt cctgaacaga tcccgggtgg
     1741 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     1801 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     1861 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     1921 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     1981 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     2041 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     2101 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     2161 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgatttta
     2221 aaataactat accagcagga ggacgtccag acacagcata ggctacctgg ccatgcccaa
     2281 ccggtgggac atttgagttg cttgcttggc actgtcctct catgcgttgg gtccactcag
     2341 tagatgcctg ttgaattggg tacgcggcca gcttggctgt ggaatgtgtg tcagttaggg
     2401 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2461 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2521 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2581 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2641 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     2701 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2761 acacgtacga ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     2821 ccccgggcag tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     2881 gtcgatccag accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     2941 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     3001 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     3061 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3121 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     3181 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3241 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3301 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3361 cccggtgcct gactgcagtg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     3421 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     3481 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     3541 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     3601 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     3661 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     3721 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3781 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3841 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3901 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3961 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4021 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4081 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4141 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4201 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4261 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4321 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4381 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4441 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4501 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4561 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4621 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4681 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4741 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4801 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4861 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4921 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4981 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5041 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5101 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5161 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5221 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5281 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5341 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5401 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5461 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5521 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5581 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5641 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5701 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5761 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5821 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     5881 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5941 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     6001 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     6061 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     6121 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     6181 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     6241 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6301 tttt
//
