LOCUS       sequence-168526-        6351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human SIRT5 with flag tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6351)
  AUTHORS   North BJ, Marshall BL, Borra MT, Denu JM, Verdin E
  TITLE     The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin 
            deacetylase.
  JOURNAL   Mol Cell. 2003 Feb . 11(2):437-44.
  PUBMED    12620231
REFERENCE   2  (bases 1 to 6351)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2003 Feb . 11(2):437-44."
COMMENT     Sequence Label: SIRT5 Flag
FEATURES             Location/Qualifiers
     source          1..6351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..80)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(993..1784,1785..1853))
                     /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     1616..1635
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1854..1958)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2033..2052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2224..2603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2604..2807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2758..2778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2852..2871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2852..2870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3873..3896
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3931..3948)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3937..4161
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4207..4635
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4294..4313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4504..4525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4644..4664)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4649..4978
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4829..4964
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4891..4910
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5045..5839
                     /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(5099..5118)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5709..5728
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6013..6134
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6050..6069)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6104..6123
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6183..6199)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6183..6199)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6196..6218)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6207..6223
                     /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(6231..6237,6238..6255,6256..6261))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6276..6297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
       61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      901 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      961 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1021 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1081 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1141 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1201 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1261 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1321 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1381 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1441 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1501 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1561 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1621 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     1681 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     1741 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1801 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1861 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1921 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1981 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     2041 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     2101 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     2161 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2221 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2281 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2341 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2401 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2461 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2521 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2581 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     2641 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     2701 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     2761 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     2821 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     2881 ctggctagcg tttaaactta agcttggtac cgagctcgga tccgaattcc atgcgacctc
     2941 tccagattgt cccaagtcga ttgatttccc agctatattg tggcctgaag cctccagcgt
     3001 ccacacgaaa ccagatttgc ctgaaaatgg ctcggccaag ttcaagtatg gcagattttc
     3061 gaaagttttt tgcaaaagca aagcacatag tcatcatctc aggagctggt gttagtgcag
     3121 aaagtggtgt tccgaccttc agaggagctg gaggttattg gagaaaatgg caagcccagg
     3181 acctggcgac tcccctggcc tttgcccaca acccgtcccg ggtgtgggag ttctaccact
     3241 accggcggga ggtcatgggg agcaaggagc ccaacgccgg gcaccgcgcc atagccgagt
     3301 gtgagacccg gctgggcaag cagggccggc gagtcgtggt catcacccag aacatcgatg
     3361 agctgcaccg caaggctggc accaagaacc ttctggagat ccatggtagc ttatttaaaa
     3421 ctcgatgtac ctcttgtgga gttgtggctg agaattacaa gagtccaatt tgtccagctt
     3481 tatcaggaaa aggtgctcca gaacctggaa ctcaagatgc cagcatccca gttgagaaac
     3541 ttccccggtg tgaagaggca ggctgcgggg gcttgctgcg acctcacgtc gtgtggtttg
     3601 gagaaaacct ggatcctgcc attctggagg aggttgacag agagctcgcc cactgtgatt
     3661 tatgtctagt ggtgggcact tcctctgtgg tgtacccagc agccatgttt gccccccagg
     3721 tggctgccag gggcgtgcca gtggctgaat ttaacacgga gaccacccca gctacgaaca
     3781 gattcaggtt tcatttccag ggaccctgtg gaacgactct tcctgaagcc cttgcctgtc
     3841 atgaaaatga aactgtttct gaattcggag gagactacaa ggacgacgat gacaagtaat
     3901 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3961 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4021 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4081 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4141 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     4201 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4261 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4321 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4381 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4441 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4501 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4561 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4621 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4681 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     4741 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4801 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4861 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     4921 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4981 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     5041 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     5101 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     5161 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     5221 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     5281 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     5341 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     5401 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     5461 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     5521 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     5581 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5641 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     5701 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     5761 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     5821 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5881 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5941 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     6001 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     6061 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6121 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6181 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6241 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6301 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct g
//
