LOCUS       sequence-483734-        7362 bp    DNA     circular SYN 18-AUG-2025
DEFINITION  Expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7362)
  AUTHORS   Michishita E, Park JY, Burneskis JM, Barrett JC, Horikawa I
  TITLE     Evolutionarily conserved and nonconserved cellular localizations and
            functions of human SIRT proteins.
  JOURNAL   Mol Biol Cell. 2005 Oct;16(10):4623-35. doi: 
            10.1091/mbc.e05-01-0033. Epub 2005 Aug 3.
  PUBMED    16079181
REFERENCE   2  (bases 1 to 7362)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1091/mbc.e05-01-0033"; journalName: "Mol Biol Cell"; date: 
            "2005-10"; volume: "16"; issue: "10"; pages: "4623-35"
COMMENT     Sequence Label: pcDNA-DEST47-SIRT3
FEATURES             Location/Qualifiers
     source          1..7362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    914..938
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2175..2199)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2210..2212,2213..2215,2216..2929)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2243..2271)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2866..2886
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2951..2968)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2957..3181
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3227..3655
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3314..3333)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3524..3545
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3664..3684)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3669..3998
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3849..3984
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3911..3930
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4065..4859
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4119..4138)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4729..4748
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5035..5156
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5072..5091)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5126..5145
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5205..5221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5205..5221)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5218..5240)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5229..5245
                     /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(5253..5259,5260..5277,5278..5283))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5298..5319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5436..5453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5607..6195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5687..5706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6366..7157,7158..7226))
                     /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     6989..7008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7227..7331)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttgat caaacaagtt tgtacaaaaa agcaggctcc gcggccgccc ccttcaccat
      961 ggcgttctgg ggttggcgcg ccgcggcagc cctccggctg tggggccggg tagttgaacg
     1021 ggtcgaggcc gggggaggcg tggggccgtt tcaggcctgc ggctgtcggc tggtgcttgg
     1081 cggcagggac gatgtgagtg cggggctgag aggcagccat ggggcccgcg gtgagccctt
     1141 ggacccggcg cgccccttgc agaggcctcc cagacccgag gtgcccaggg cattccggag
     1201 gcagccgagg gcagcagctc ccagtttctt cttttcgagt attaaaggtg gaagaaggtc
     1261 catatctttt tctgtgggtg cttcaagtgt tgttggaagt ggaggcagca gtgacaaggg
     1321 gaagctttcc ctgcaggatg tagctgagct gattcgggcc agagcctgcc agagggtggt
     1381 ggtcatggtg ggggccggca tcagcacacc cagtggcatt ccagacttca gatcgccggg
     1441 gagtggcctg tacagcaacc tccagcagta cgatctcccg taccccgagg ccatttttga
     1501 actcccattc ttctttcaca accccaagcc ctttttcact ttggccaagg agctgtaccc
     1561 tggaaactac aagcccaacg tcactcacta ctttctccgg ctgcttcatg acaaggggct
     1621 gcttctgcgg ctctacacgc agaacatcga tgggcttgag agagtgtcgg gcatccctgc
     1681 ctcaaagctg gttgaagctc atggaacctt tgcctctgcc acctgcacag tctgccaaag
     1741 acccttccca ggggaggaca ttcgggctga cgtgatggca gacagggttc cccgctgccc
     1801 ggtctgcacc ggcgttgtga agcccgacat tgtgttcttt ggggagccgc tgccccagag
     1861 gttcttgctg catgtggttg atttccccat ggcagatctg ctgctcatcc ttgggacctc
     1921 cctggaggtg gagccttttg ccagcttgac cgaggccgtg cggagctcag ttccccgact
     1981 gctcatcaac cgggacttgg tggggccctt ggcttggcat cctcgcagca gggacgtggc
     2041 ccagctgggg gacgtggttc acggcgtgga aagcctagtg gagcttctgg gctggacaga
     2101 agagatgcgg gaccttgtgc agcgggaaac tgggaagctt gatggaccag acaaatttaa
     2161 gggtgggcgc gccgacccag ctttcttgta caaagtggtt cgatctagaa tggctagcaa
     2221 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
     2281 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gctacatacg gaaagcttac
     2341 ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac ttgtcactac
     2401 tttctcttat ggtgttcaat gcttttcccg ttatccggat catatgaaac ggcatgactt
     2461 tttcaagagt gccatgcccg aaggttatgt acaggaacgc actatatctt tcaaagatga
     2521 cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat
     2581 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt ctcggacaca aactcgagta
     2641 caactataac tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc
     2701 taacttcaaa attcgccaca acattgaaga tggatccgtt caactagcag accattatca
     2761 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtcgac
     2821 acaatctgcc ctttcgaaag atcccaacga aaagcgtgac cacatggtcc ttcttgagtt
     2881 tgtaactgct gctgggatta cacatggcat ggatgagctc tacaaataat gaattaaacc
     2941 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3001 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3061 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3121 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3181 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3241 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3301 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3361 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3421 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3481 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3541 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3601 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3661 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3721 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3781 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3841 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3901 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3961 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     4021 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4081 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4141 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4201 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4261 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4321 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4381 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4441 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4501 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4561 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4621 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4681 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4741 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4801 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4861 cgggactctg gggttcgcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4921 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4981 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     5041 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     5101 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     5161 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     5221 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5281 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5341 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5401 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5461 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5521 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5581 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5641 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5701 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5761 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5821 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5881 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5941 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6001 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6061 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6121 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6181 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6241 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6301 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6361 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6421 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6481 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6541 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6601 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6661 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6721 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6781 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6841 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6901 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6961 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7021 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7081 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7141 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7201 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7261 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7321 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
