LOCUS       sequence-483745-        7332 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 7332)
  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 7332)
  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-SIRT2
FEATURES             Location/Qualifiers
     source          1..7332
                     /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(2145..2169)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2180..2182,2183..2185,2186..2899)
                     /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(2213..2241)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2836..2856
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2921..2938)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2927..3151
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3197..3625
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3284..3303)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3494..3515
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3634..3654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3639..3968
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3819..3954
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3881..3900
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4035..4829
                     /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(4089..4108)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4699..4718
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5005..5126
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5042..5061)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5096..5115
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5175..5191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5175..5191)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5188..5210)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5199..5215
                     /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(5223..5229,5230..5247,5248..5253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5268..5289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5406..5423)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5577..6165)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5657..5676)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6336..7127,7128..7196))
                     /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     6959..6978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7197..7301)
                     /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 ggcagagcca gacccctctc accctctgga gacccaggca gggaaggtgc aggaggctca
     1021 ggactcagat tcagactctg agggaggagc cgctggtgga gaagcagaca tggacttcct
     1081 gcggaactta ttctcccaga cgctcagcct gggcagccag aaggagcgtc tgctggacga
     1141 gctgaccttg gaaggggtgg cccggtacat gcagagcgaa cgctgtcgca gagtcatctg
     1201 tttggtggga gctggaatct ccacatccgc aggcatcccc gactttcgct ctccatccac
     1261 cggcctctat gacaacctag agaagtacca tcttccctac ccagaggcca tctttgagat
     1321 cagctatttc aagaaacatc cggaaccctt cttcgccctc gccaaggaac tctatcctgg
     1381 gcagttcaag ccaaccatct gtcactactt catgcgcctg ctgaaggaca aggggctact
     1441 cctgcgctgc tacacgcaga acatagatac cctggagcga atagccgggc tggaacagga
     1501 ggacttggtg gaggcgcacg gcaccttcta cacatcacac tgcgtcagcg ccagctgccg
     1561 gcacgaatac ccgctaagct ggatgaaaga gaagatcttc tctgaggtga cgcccaagtg
     1621 tgaagactgt cagagcctgg tgaagcctga tatcgtcttt tttggtgaga gcctcccagc
     1681 gcgtttcttc tcctgtatgc agtcagactt cctgaaggtg gacctcctcc tggtcatggg
     1741 tacctccttg caggtgcagc cctttgcctc cctcatcagc aaggcacccc tctccacccc
     1801 tcgcctgctc atcaacaagg agaaagctgg ccagtcggac cctttcctgg ggatgattat
     1861 gggcctcgga ggaggcatgg actttgactc caagaaggcc tacagggacg tggcctggct
     1921 gggtgaatgc gaccagggct gcctggccct tgctgagctc cttggatgga agaaggagct
     1981 ggaggacctt gtccggaggg agcacgccag catagatgcc cagtcggggg cgggggtccc
     2041 caaccccagc acttcagctt cccccaagaa gtccccgcca cctgccaagg acgaggccag
     2101 gacaacagag agggagaaac cccagtttaa gggtgggcgc gccgacccag ctttcttgta
     2161 caaagtggtt cgatctagaa tggctagcaa aggagaagaa cttttcactg gagttgtccc
     2221 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg
     2281 tgaaggtgat gctacatacg gaaagcttac ccttaaattt atttgcacta ctggaaaact
     2341 acctgttcca tggccaacac ttgtcactac tttctcttat ggtgttcaat gcttttcccg
     2401 ttatccggat catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt
     2461 acaggaacgc actatatctt tcaaagatga cgggaactac aagacgcgtg ctgaagtcaa
     2521 gtttgaaggt gatacccttg ttaatcgtat cgagttaaaa ggtattgatt ttaaagaaga
     2581 tggaaacatt ctcggacaca aactcgagta caactataac tcacacaatg tatacatcac
     2641 ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attcgccaca acattgaaga
     2701 tggatccgtt caactagcag accattatca acaaaatact ccaattggcg atggccctgt
     2761 ccttttacca gacaaccatt acctgtcgac acaatctgcc ctttcgaaag atcccaacga
     2821 aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat
     2881 ggatgagctc tacaaataat gaattaaacc cgctgatcag cctcgactgt gccttctagt
     2941 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3001 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3061 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3121 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3181 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3241 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3301 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3361 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3421 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3481 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3541 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3601 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3661 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3721 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3781 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3841 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3901 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3961 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     4021 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4081 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4141 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4201 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4261 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4321 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4381 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4441 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4501 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4561 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4621 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4681 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4741 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4801 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac
     4861 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4921 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4981 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     5041 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5101 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5161 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     5221 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     5281 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5341 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     5401 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5461 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5521 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5581 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5641 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5701 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5761 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5821 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5881 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5941 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6001 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6061 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6121 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6181 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6241 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6301 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6361 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6421 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6481 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6541 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6601 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6661 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6721 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6781 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6841 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6901 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6961 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7021 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7081 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7141 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7201 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7261 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7321 ccacctgacg tc
//
