LOCUS       sequence-483732-        7095 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 7095)
  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 7095)
  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-SIRT5
FEATURES             Location/Qualifiers
     source          1..7095
                     /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(1908..1932)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1943..1945,1946..1948,1949..2662)
                     /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(1976..2004)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2599..2619
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2684..2701)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2690..2914
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2960..3388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3047..3066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3257..3278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3397..3417)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3402..3731
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3582..3717
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3644..3663
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3798..4592
                     /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(3852..3871)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4462..4481
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4768..4889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4805..4824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4859..4878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4938..4954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4938..4954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4951..4973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4962..4978
                     /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(4986..4992,4993..5010,5011..5016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5031..5052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5169..5186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5340..5928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5420..5439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6099..6890,6891..6959))
                     /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     6722..6741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6960..7064)
                     /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 gcgacctctc cagattgtcc caagtcgatt gatttcccag ctatattgtg gcctgaagcc
     1021 tccagcgtcc acacgaaacc agatttgcct gaaaatggct cggccaagtt caagtatggc
     1081 agattttcga aagttttttg caaaagcaaa gcacatagtc atcatctcag gagctggtgt
     1141 tagtgcagaa agtggtgttc cgaccttcag aggagctgga ggttattgga gaaaatggca
     1201 agcccaggac ctggcgactc ccctggcctt tgcccacaac ccgtcccggg tgtgggagtt
     1261 ctaccactac cggcgggagg tcatggggag caaggagccc aacgccgggc accgcgccat
     1321 agccgagtgt gagacccggc tgggcaagca gggccggcgg gtcgtggtca tcacccagaa
     1381 catcgatgag ctgcaccgca aggctggcac caagaacctt ctggagatcc atggtagctt
     1441 atttaaaact cgatgtacct cttgtggagt tgtggctgag aattacaaga gtccaatttg
     1501 tccagcttta tcaggaaaag gtgctccaga acctggaact caagatgcca gcatcccagt
     1561 tgagaaactt ccccggtgtg aagaggcagg ctgcgggggc ttgctgcgac ctcacgtcgt
     1621 gtggtttgga gaaaacctgg atcctgccat tctggaggag gttgacagag agctcgccca
     1681 ctgtgattta tgtctagtgg tgggcacttc ctctgtggtg tacccagcag ccatgtttgc
     1741 cccccaggtg gctgccaggg gcgtgccagt ggctgaattt aacacggaga ccaccccagc
     1801 tacgaacaga ttcaggtttc atttccaggg accctgtgga acgactcttc ctgaagccct
     1861 tgcctgtcat gaaaatgaaa ctgtttcttt taagggtggg cgcgccgacc cagctttctt
     1921 gtacaaagtg gttcgatcta gaatggctag caaaggagaa gaacttttca ctggagttgt
     1981 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
     2041 gggtgaaggt gatgctacat acggaaagct tacccttaaa tttatttgca ctactggaaa
     2101 actacctgtt ccatggccaa cacttgtcac tactttctct tatggtgttc aatgcttttc
     2161 ccgttatccg gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta
     2221 tgtacaggaa cgcactatat ctttcaaaga tgacgggaac tacaagacgc gtgctgaagt
     2281 caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aaaggtattg attttaaaga
     2341 agatggaaac attctcggac acaaactcga gtacaactat aactcacaca atgtatacat
     2401 cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacattga
     2461 agatggatcc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc
     2521 tgtcctttta ccagacaacc attacctgtc gacacaatct gccctttcga aagatcccaa
     2581 cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg
     2641 catggatgag ctctacaaat aatgaattaa acccgctgat cagcctcgac tgtgccttct
     2701 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2761 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2821 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2881 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2941 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3001 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3061 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3121 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3181 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3241 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3301 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3361 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3421 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3481 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3541 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3601 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3661 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3721 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3781 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3841 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3901 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3961 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4021 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4081 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4141 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4201 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4261 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4321 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4381 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4441 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4501 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4561 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg cgaaatgacc
     4621 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4681 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4741 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4801 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4861 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4921 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4981 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     5041 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5101 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5161 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5221 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5281 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5341 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5401 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5461 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5521 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5581 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5641 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5701 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5761 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5821 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5881 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5941 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6001 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6061 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6121 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6181 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6241 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6301 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6361 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6421 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6481 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6541 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6601 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6661 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6721 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6781 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6841 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6901 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6961 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7021 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7081 gtgccacctg acgtc
//
