LOCUS       40924_835        5420 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Mammalian expression of flag-tagged SIRT1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5420)
  AUTHORS   Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H,
            Ross SE, Mostoslavsky R, Cohen HY, Hu LS, Cheng HL, Jedrychowski MP,
            Gygi SP, Sinclair DA, Alt FW, Greenberg ME
  TITLE     Stress-dependent regulation of FOXO transcription factors by the 
            SIRT1 deacetylase.
  JOURNAL   Science 2004 Mar 26;303(5666):2011-5. Epub 2004 Feb 19.
  PUBMED    14976264
REFERENCE   2  (bases 1 to 5420)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5420)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science"; 
            date: "2004-03-26"; volume: "303(5666)"; pages: "2011-5. Epub 2004
            Feb 19"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(55..75)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        73..362
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             404..427
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             440..2677
                     /codon_start=1
                     /label=SIRT1
                     /note="human NAD+-dependent protein deacetylase sirtuin-1"
                     /translation="ADEAALALQPGGSPSAAGADREAASSPAGEPLRKRPRRDGPGLER
                     SPGEPGGAAPEREVPAAARGCPGAAAAALWREAEAEAAAAGGEQEAQATAAAGEGDNGP
                     GLQGPSREPPLADNLYDEDDDDEGEEEEEAAAAAIGYRDNLLFGDEIITNGFHSCESDE
                     EDRASHASSSDWTPRPRIGPYTFVQQHLMIGTDPRTILKDLLPETIPPPELDDMTLWQI
                     VINILSEPPKRKKRKDINTIEDAVKLLQECKKIIVLTGAGVSVSCGIPDFRSRDGIYAR
                     LAVDFPDLPDPQAMFDIEYFRKDPRPFFKFAKEIYPGQFQPSLCHKFIALSDKEGKLLR
                     NYTQNIDTLEQVAGIQRIIQCHGSFATASCLICKYKVDCEAVRGDIFNQVVPRCPRCPA
                     DEPLAIMKPEIVFFGENLPEQFHRAMKYDKDEVDLLIVIGSSLKVRPVALIPSSIPHEV
                     PQILINREPLPHLHFDVELLGDCDVIINELCHRLGGEYAKLCCNPVKLSEITEKPPRTQ
                     KELAYLSELPPTPLHVSEDSSSPERTSPPDSSVIVTLLDQAAKSNDDLDVSESKGCMEE
                     KPQEVQTSRNVESIAEQMENPDLKNVGSSTGEKNERTSVAGTVRKCWPNRVAKEQISRR
                     LDGNQYLFLPPNRYIFHGAEVYSDSEDDVLSSSSCGSNSDSGTCQSPSLEEPMEDESEI
                     EEFYNGLEDEPDVPERAGGAGFGTDGDDQEAINEAISVKQEVTDMNYPSNKS"
     promoter        complement(2756..2774)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2781..2797)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3048..3182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3241..3260)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     primer_bind     complement(3525..3542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3696..4284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4458..5315)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5316..5420)
                     /label=AmpR promoter
ORIGIN
        1 cacatttccc cgaaaagtgc cacctgacct tctgaggcgg aaagaaccag ctgtggaatg
       61 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
      121 tgcatctcaa ttagtcagca aggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
      181 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
      241 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
      301 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgaag aggctttttt
      361 ggaggagatc taagcttggt accgagctcg gatcctcagg atggactaca aggacgatga
      421 cgataaaccc gggaattcgg cggacgaggc ggccctcgcc cttcagcccg gcggctcccc
      481 ctcggcggcg ggggccgaca gggaggccgc gtcgtccccc gccggggagc cgctccgcaa
      541 gaggccgcgg agagatggtc ccggcctcga gcggagcccg ggcgagcccg gtggggcggc
      601 cccagagcgt gaggtgccgg cggcggccag gggctgcccg ggtgcggcgg cggcggcgct
      661 gtggcgggag gcggaggcag aggcggcggc ggcaggcggg gagcaagagg cccaggcgac
      721 tgcggcggct ggggaaggag acaatgggcc gggcctgcag ggcccatctc gggagccacc
      781 gctggccgac aacttgtacg acgaagacga cgacgacgag ggcgaggagg aggaagaggc
      841 ggcggcggcg gcgattgggt accgagataa ccttctgttc ggtgatgaaa ttatcactaa
      901 tggttttcat tcctgtgaaa gtgatgagga ggatagagcc tcacatgcaa gctctagtga
      961 ctggactcca aggccacgga taggtccata tacttttgtt cagcaacatc ttatgattgg
     1021 cacagatcct cgaacaattc ttaaagattt attgccggaa acaatacctc cacctgagtt
     1081 ggatgatatg acactgtggc agattgttat taatatcctt tcagaaccac caaaaaggaa
     1141 aaaaagaaaa gatattaata caattgaaga tgctgtgaaa ttactgcaag agtgcaaaaa
     1201 aattatagtt ctaactggag ctggggtgtc tgtttcatgt ggaatacctg acttcaggtc
     1261 aagggatggt atttatgctc gccttgctgt agacttccca gatcttccag atcctcaagc
     1321 gatgtttgat attgaatatt tcagaaaaga tccaagacca ttcttcaagt ttgcaaagga
     1381 aatatatcct ggacaattcc agccatctct ctgtcacaaa ttcatagcct tgtcagataa
     1441 ggaaggaaaa ctacttcgca actataccca gaacatagac acgctggaac aggttgcggg
     1501 aatccaaagg ataattcagt gtcatggttc ctttgcaaca gcatcttgcc tgatttgtaa
     1561 atacaaagtt gactgtgaag ctgtacgagg agatattttt aatcaggtag ttcctcgatg
     1621 tcctaggtgc ccagctgatg aaccgcttgc tatcatgaaa ccagagattg tgttttttgg
     1681 tgaaaattta ccagaacagt ttcatagagc catgaagtat gacaaagatg aagttgacct
     1741 cctcattgtt attgggtctt ccctcaaagt aagaccagta gcactaattc caagttccat
     1801 accccatgaa gtgcctcaga tattaattaa tagagaacct ttgcctcatc tgcattttga
     1861 tgtagagctt cttggagact gtgatgtcat aattaatgaa ttgtgtcata ggttaggtgg
     1921 tgaatatgcc aaactttgct gtaaccctgt aaagctttca gaaattactg aaaaacctcc
     1981 acgaacacaa aaagaattgg cttatttgtc agagttgcca cccacacctc ttcatgtttc
     2041 agaagactca agttcaccag aaagaacttc accaccagat tcttcagtga ttgtcacact
     2101 tttagaccaa gcagctaaga gtaatgatga tttagatgtg tctgaatcaa aaggttgtat
     2161 ggaagaaaaa ccacaggaag tacaaacttc taggaatgtt gaaagtattg ctgaacagat
     2221 ggaaaatccg gatttgaaga atgttggttc tagtactggg gagaaaaatg aaagaacttc
     2281 agtggctgga acagtgagaa aatgctggcc taatagagtg gcaaaggagc agattagtag
     2341 gcggcttgat ggtaatcagt atctgttttt gccaccaaat cgttacattt tccatggcgc
     2401 tgaggtatat tcagactctg aagatgacgt cttatcctct agttcttgtg gcagtaacag
     2461 tgatagtggg acatgccaga gtccaagttt agaagaaccc atggaggatg aaagtgaaat
     2521 tgaagaattc tacaatggct tagaagatga gcctgatgtt ccagagagag ctggaggagc
     2581 tggatttggg actgatggag atgatcaaga ggcaattaat gaagctatat ctgtgaaaca
     2641 ggaagtaaca gacatgaact atccatcaaa caaatcatag tgtaataatt gtgcaggtac
     2701 aggaattgtt ccactctaga caagggcgaa ttcgcggccg ctaaattcaa ttcgccctat
     2761 agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     2821 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     2881 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctatacct agataagtaa
     2941 tgatcataat cagccatatc acatctgtag aggttttact tgctttaaaa aacctcccac
     3001 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3061 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3121 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     3181 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     3241 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     3301 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     3361 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     3421 gcttcctaat gcaggagtcg cataagggag agcgtcgata tgcggtgtga aataccgcac
     3481 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     3541 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3601 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3661 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3721 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3781 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3841 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     3901 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     3961 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4021 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4081 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     4141 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4201 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4261 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4321 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4381 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4441 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4501 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4561 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4621 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4681 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4741 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     4801 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4861 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     4921 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     4981 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5041 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     5101 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5161 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5221 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5281 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5341 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5401 aaacaaatag gggttccgcg
//
