LOCUS       sequence-483733-        7230 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 7230)
  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 7230)
  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-SIRT6
FEATURES             Location/Qualifiers
     source          1..7230
                     /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(2043..2067)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2078..2080,2081..2083,2084..2797)
                     /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(2111..2139)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2734..2754
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2819..2836)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2825..3049
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3095..3523
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3182..3201)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3392..3413
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3532..3552)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3537..3866
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3717..3852
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3779..3798
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3933..4727
                     /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(3987..4006)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4597..4616
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4903..5024
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4940..4959)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4994..5013
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5073..5089)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5073..5089)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5086..5108)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5097..5113
                     /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(5121..5127,5128..5145,5146..5151))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5166..5187
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5304..5321)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5475..6063)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5555..5574)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6234..7025,7026..7094))
                     /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     6857..6876
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7095..7199)
                     /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 gtcggtgaat tacgcggcgg ggctgtcgcc gtacgcggac aagggcaagt gcggcctccc
     1021 ggagatcttc gaccccccgg aggagctgga gcggaaggtg tgggaactgg cgaggctggt
     1081 ctggcagtct tccagtgtgg tgttccacac gggtgccggc atcagcactg cctctggcat
     1141 ccccgacttc aggggtcccc acggagtctg gaccatggag gagcgaggtc tggcccccaa
     1201 gttcgacacc acctttgaga gcgcgcggcc cacgcagacc cacatggcgc tggtgcagct
     1261 ggagcgcgtg ggcctcctcc gcttcctggt cagccagaac gtggacgggc tccatgtgcg
     1321 ctcaggcttc cccagggaca aactggcaga gctccacggg aacatgtttg tggaagaatg
     1381 tgccaagtgt aagacgcagt acgtccgaga cacagtcgtg ggcaccatgg gcctgaaggc
     1441 cacgggccgg ctctgcaccg tggctaaggc aagggggctg cgagcctgca ggggagagct
     1501 gagggacacc atcctagact gggaggactc cctgcccgac cgggacctgg cactcgccga
     1561 tgaggccagc aggaacgccg acctgtccat cacgctgggt acatcgctgc agatccggcc
     1621 cagcgggaac ctgccgctgg ctaccaagcg ccggggaggc cgcctggtca tcgtcaacct
     1681 gcagcccacc aagcacgacc gccatgctga cctccgcatc catggctacg ttgacgaggt
     1741 catgacccgg ctcatgaagc acctggggct ggagatcccc gcctgggacg gcccccgtgt
     1801 gctggagagg gcgctgccac ccctgccccg cccgcccacc cccaagctgg agcccaagga
     1861 ggaatctccc acccggatca acggctctat ccccgccggc cccaagcagg agccctgcgc
     1921 ccagcacaac ggctcagagc ccgccagccc caaacgggag cggcccacca gccctgcccc
     1981 ccacagaccc cccaaaaggg tgaaggccaa ggcggtcccc agctttaagg gtgggcgcgc
     2041 cgacccagct ttcttgtaca aagtggttcg atctagaatg gctagcaaag gagaagaact
     2101 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     2161 ttctgtcagt ggagagggtg aaggtgatgc tacatacgga aagcttaccc ttaaatttat
     2221 ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tctcttatgg
     2281 tgttcaatgc ttttcccgtt atccggatca tatgaaacgg catgactttt tcaagagtgc
     2341 catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaactacaa
     2401 gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg
     2461 tattgatttt aaagaagatg gaaacattct cggacacaaa ctcgagtaca actataactc
     2521 acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat
     2581 tcgccacaac attgaagatg gatccgttca actagcagac cattatcaac aaaatactcc
     2641 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgccct
     2701 ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc
     2761 tgggattaca catggcatgg atgagctcta caaataatga attaaacccg ctgatcagcc
     2821 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2881 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2941 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3001 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3061 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3121 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3181 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3241 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3301 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3361 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3421 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3481 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     3541 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3601 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3661 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3721 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3781 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     3841 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     3901 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3961 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4021 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4081 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4141 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4201 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4261 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4321 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4381 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4441 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4501 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4561 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4621 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4681 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4741 gttcgcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4801 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4861 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4921 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4981 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     5041 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5101 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5161 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5221 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5281 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5341 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5401 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5461 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5521 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5581 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5641 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5701 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5761 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5821 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5881 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5941 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6001 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6061 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6121 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6181 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6241 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6301 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6361 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6421 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6481 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6541 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6601 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6661 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6721 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6781 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6841 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6901 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6961 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7021 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7081 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7141 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7201 acatttcccc gaaaagtgcc acctgacgtc
//
