LOCUS       sequence-488638-        6598 bp    DNA     circular SYN 07-OCT-2025
DEFINITION  mammalian expression of ATP6 with mitochondrial targeting sequence, 
            Myc, and FLAG tags.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  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 6598)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 7, 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-G1-ATP6-Myc-FLAG (puromycin)
FEATURES             Location/Qualifiers
     source          1..6598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     610..629
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        610..628
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1569..1598
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1617..1640
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1660..1676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1687..2309
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1886..1905)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2333..2353)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2338..2667
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2518..2653
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2725..3324
                     /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(2725..2744)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3221..3241
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3503..3624
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3540..3559)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3594..3613
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3673..3689)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3686..3708)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3697..3713
                     /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(3721..3727,3728..3745,3746..3751))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3766..3787
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3904..3921)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4075..4663)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4155..4174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4834..5625,5626..5694))
                     /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     5457..5476
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5695..5799)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5826..6281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5913..5932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6123..6144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6407..6429
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6421..6438
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6422..6438
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agaattttgt aatacgactc actatagggc ggccgggaat tcgtcgactg gatccggtac
      661 cgaggagatc tgccgccgcg atcgccatgc agaccgccgg ggcattattc atttctccag
      721 ctctgatccg ctgttgtacc aggggtctaa tcaggcctgt gtctgcctcc ttcttgaata
      781 gcccagtgaa ttcatctaaa cagccttcct acagcaactt cccactccag gtggccagac
      841 gggagttcca gaccagtgtt gtctcccggg acattgacac ggcgacgcgt aacgaaaatc
      901 tgttcgcttc attcattgcc cccacaatcc taggcctacc cgccgcagta ctgatcattc
      961 tatttccccc tctattgatc cccacctcca aatatctcat caacaaccga ctaatcacca
     1021 cccaacaatg gctaatcaaa ctaacctcaa aacaaatgat gaccatgcac aacactaaag
     1081 gacgaacctg gtctcttatg ctagtatcct taatcatttt tattgccaca actaacctcc
     1141 tcggactcct gcctcactca tttacaccaa ccacccaact atctatgaac ctagccatgg
     1201 ccatcccctt atgggcgggc acagtgatta tgggctttcg ctctaagatt aaaaatgccc
     1261 tagcccactt cttaccacaa ggcacaccta caccccttat ccccatgcta gttattatcg
     1321 aaaccatcag cctactcatt caaccaatgg ccctggccgt acgcctaacc gctaacatta
     1381 ctgcaggcca cctactcatg cacctaattg gaagcgccac cctagcaatg tcaaccatta
     1441 accttccctc tacacttatc atcttcacaa ttctaattct actgactatc ctagaaatcg
     1501 ctgtcgcctt aatccaagcc tacgttttca cacttctagt aagcctctac ctgcacgaca
     1561 acacactcga gcagaaactc atctcagaag aggatctggc agcaaatgat atcctggatt
     1621 acaaggatga cgacgataag gtttaaacgg ccggccgcgg tcatagctgt ttcctgaaca
     1681 gatcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
     1741 actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag
     1801 gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg
     1861 aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa
     1921 tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc
     1981 gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag
     2041 agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac
     2101 ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc
     2161 cttctgattt taaaataact ataccagcag gaggacgtcc agacacagca taggctacct
     2221 ggccatgccc aaccggtggg acatttgagt tgcttgcttg gcactgtcct ctcatgcgtt
     2281 gggtccactc agtagatgcc tgttgaattg ggtacgcggc cagcttggct gtggaatgtg
     2341 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2401 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2461 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     2521 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     2581 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     2641 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     2701 tctgatcaag agacacgtac gaccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2761 cgcgacgacg tcccccgggc agtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     2821 acgcgccaca ccgtcgatcc agaccgccac atcgagcggg tcaccgagct gcaagaactc
     2881 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     2941 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3001 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3061 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     3121 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     3181 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     3241 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     3301 atgacccgca agcccggtgc ctgactgcag tgggactctg gggttcgaaa tgaccgacca
     3361 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     3421 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     3481 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     3541 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     3601 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     3661 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3721 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3781 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3841 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3901 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3961 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4021 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4081 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4141 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4201 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4261 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     4321 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     4381 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4441 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4501 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4561 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4621 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4681 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4741 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4801 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4861 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4921 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     4981 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5041 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5101 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5161 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5221 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     5281 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     5341 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     5401 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5461 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5521 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5581 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5641 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5701 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5761 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5821 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5881 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     5941 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6001 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     6061 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     6121 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     6181 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     6241 atttaacgcg aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg
     6301 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     6361 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     6421 tgtaaaacga cggccagtgc caagctgatc tatacattga atcaatattg gcaattagcc
     6481 atattagtca ttggttatat agcataaatc aatattggct attggccatt gcatacgttg
     6541 tatctatatc ataatatgta catttatatt ggctcatgtc caatatgacc gccatgtt
//
