LOCUS       sequence-483736-        7768 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 7768)
  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 7768)
  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-DEST40-SIRT1
FEATURES             Location/Qualifiers
     source          1..7768
                     /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    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             956..3196
                     /codon_start=1
                     /product="human NAD+-dependent protein deacetylase 
                     sirtuin-1"
                     /label=SIRT1
                     /note="homolog of budding yeast Sir2"
                     /translation="MADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLE
                     RSPGEPGGAAPEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNG
                     PGLQGPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDEIITNGFHSCESD
                     EEDRASHASSSDWTPRPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLWQ
                     IVINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIYA
                     RLAVDFPDLPDPQAMFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLL
                     RNYTQNIDTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGDIFNQVVPRCPRCP
                     ADEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHE
                     VPQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSEITEKPPRT
                     QKELAYLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCME
                     EKPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISR
                     RLDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPMEDESE
                     IEEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEAISVKQEVTDMNYPSNKS"
     protein_bind    complement(3216..3240)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3266..3307
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3317..3334
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3357..3374)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3363..3587
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3633..4061
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3720..3739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3930..3951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4070..4090)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4075..4404
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4255..4390
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4317..4336
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4471..5265
                     /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(4525..4544)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5135..5154
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5441..5562
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5478..5497)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5532..5551
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5611..5627)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5611..5627)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5624..5646)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5635..5651
                     /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(5659..5665,5666..5683,5684..5689))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5704..5725
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5842..5859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6013..6601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6093..6112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6772..7563,7564..7632))
                     /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     7395..7414
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7633..7737)
                     /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 taagctatca acaagtttgt acaaaaaagc aggctccgcg gccgccccct tcaccatggc
      961 ggacgaggcg gccctcgccc ttcagcccgg cggctccccc tcggcggcgg gggccgacag
     1021 ggaggccgcg tcgtcccccg ccggggagcc gctccgcaag aggccgcgga gagatggtcc
     1081 cggcctcgag cggagcccgg gcgagcccgg tggggcggcc ccagagcgtg aggtgccggc
     1141 ggcggccagg ggctgcccgg gtgcggcggc ggcggcgctg tggcgggagg cggaggcaga
     1201 ggcggcggcg gcaggcgggg agcaagaggc ccaggcgact gcggcggctg gggaaggaga
     1261 caatgggccg ggcctgcagg gcccatctcg ggagccaccg ctggccgaca acttgtacga
     1321 cgaagacgac gacgacgagg gcgaggagga ggaagaggcg gcggcggcgg cgattgggta
     1381 ccgagataac cttctgttcg gtgatgaaat tatcactaat ggttttcatt cctgtgaaag
     1441 tgatgaggag gatagagcct cacatgcaag ctctagtgac tggactccaa ggccacggat
     1501 aggtccatat acttttgttc agcaacatct tatgattggc acagatcctc gaacaattct
     1561 taaagattta ttgccggaaa caatacctcc acctgagttg gatgatatga cactgtggca
     1621 gattgttatt aatatccttt cagaaccacc aaaaaggaaa aaaagaaaag atattaatac
     1681 aattgaagat gctgtgaaat tactgcaaga gtgcaaaaaa attatagttc taactggagc
     1741 tggggtgtct gtttcatgtg gaatacctga cttcaggtca agggatggta tttatgctcg
     1801 ccttgctgta gacttcccag atcttccaga tcctcaagcg atgtttgata ttgaatattt
     1861 cagaaaagat ccaagaccat tcttcaagtt tgcaaaggaa atatatcctg gacaattcca
     1921 gccatctctc tgtcacaaat tcatagcctt gtcagataag gaaggaaaac tacttcgcaa
     1981 ctatacccag aacatagaca cgctggaaca ggttgcggga atccaaagga taattcagtg
     2041 tcatggttcc tttgcaacag catcttgcct gatttgtaaa tacaaagttg actgtgaagc
     2101 tgtacgagga gatattttta atcaggtagt tcctcgatgt cctaggtgcc cagctgatga
     2161 accgcttgct atcatgaaac cagagattgt gttttttggt gaaaatttac cagaacagtt
     2221 tcatagagcc atgaagtatg acaaagatga agttgacctc ctcattgtta ttgggtcttc
     2281 cctcaaagta agaccagttg cactaattcc aagttccata ccccatgaag tgcctcagat
     2341 attaattaat agagaacctt tgcctcatct gcattttgat gtagagcttc ttggagactg
     2401 tgatgtcata attaatgaat tgtgtcatag gttaggtggt gaatatgcca aactttgctg
     2461 taaccctgta aagctttcag aaattactga aaaacctcca cgaacacaaa aagaattggc
     2521 ttatttgtca gagttgccac ccacacctct tcatgtttca gaagactcaa gttcaccaga
     2581 aagaacttca ccaccagatt cttcagtgat tgtcacactt ttagaccaag cagctaagag
     2641 taatgatgat ttagatgtgt ctgaatcaaa aggttgtatg gaagaaaaac cacaggaagt
     2701 acaaacttct aggaatgttg aaagtattgc tgaacagatg gaaaatccgg atttgaagaa
     2761 tgttggttct agtactgggg agaaaaatga aagaacttca gtggctggaa cagtgagaaa
     2821 atgctggcct aatagagtgg caaaggagca gattagtagg cggcttgatg gtaatcagta
     2881 tctgtttttg ccaccaaatc gttacatttt ccatggcgct gaggtatatt cagactctga
     2941 agatgacgtc ttatcctcta gttcttgtgg cagtaacagt gatagtggga catgccagag
     3001 tccaagttta gaagagccca tggaggatga aagtgaaatt gaagaattct acaatggctt
     3061 agaagatgag cctgatgttc cagagagagc tggaggagct ggatttggga ctgatggaga
     3121 tgatcaagag gcaattaatg aagctatatc tgtgaaacag gaagtaacag acatgaacta
     3181 tccatcaaac aaatcattta agggtgggcg cgccgaccca gctttcttgt acaaagtggt
     3241 tgatctagag ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc tcggtctcga
     3301 ttctacgcgt accggtcatc atcaccatca ccattgagtt taaacccgct gatcagcctc
     3361 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3421 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3481 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3541 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3601 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3661 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3721 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3781 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3841 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3901 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3961 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4021 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     4081 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4141 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4201 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4261 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4321 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4381 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4441 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4501 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4561 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4621 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4681 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4741 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4801 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4861 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4921 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4981 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     5041 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5101 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5161 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5221 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5281 tcgcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     5341 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5401 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5461 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5521 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5581 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5641 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5701 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5761 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5821 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5881 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5941 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6001 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6061 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6121 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6181 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6241 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6301 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6361 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6421 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6481 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6541 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6601 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6661 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6721 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6781 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6841 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6901 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6961 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7021 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7081 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7141 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7201 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7261 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7321 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7381 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7441 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7501 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7561 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7621 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7681 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7741 atttccccga aaagtgccac ctgacgtc
//
