LOCUS       sequence-364548-        6618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human SIRT3 with flag tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6618)
  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 6618)
  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: SIRT3 Flag
FEATURES             Location/Qualifiers
     source          1..6618
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..33)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        18..347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      198..333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     260..279
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             414..1208
                     /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(468..487)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1078..1097
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1382..1503
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1419..1438)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1473..1492
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1552..1568)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1552..1568)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1565..1587)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1576..1592
                     /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(1600..1606,1607..1624,1625..1630))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1645..1666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1783..1800)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1954..2542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2034..2053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2713..3504,3505..3573))
                     /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     3336..3355
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3574..3678)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3753..3772)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3944..4323
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4324..4527
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4478..4498
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4572..4591
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4572..4590
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5860..5883
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5918..5935)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5924..6148
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6194..6618,1..4)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6281..6300)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6491..6512
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
       61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      361 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
      421 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
      481 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
      541 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
      601 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
      661 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
      721 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
      781 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
      841 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
      901 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
      961 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     1021 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     1081 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     1141 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     1201 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     1261 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     1321 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     1381 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1441 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1501 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     1561 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1621 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1681 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     1741 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1801 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1861 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1921 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1981 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2041 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2101 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2161 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2221 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2281 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2341 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2401 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2461 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2521 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2581 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2641 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2701 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2761 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2821 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2881 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2941 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3001 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3061 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3121 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3181 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3241 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3301 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3361 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3421 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3481 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3541 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3601 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3661 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     3721 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3781 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     3841 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     3901 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
     3961 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     4021 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     4081 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     4141 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     4201 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     4261 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     4321 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     4381 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     4441 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     4501 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
     4561 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta
     4621 agcttggtac cgagctcgga tccgaattcc atggcgttct ggggttggcg cgccgcggca
     4681 gccctccggc tgtggggccg ggtagttgaa cgggtcgagg ccgggggagg cgtggggccg
     4741 tttcaggcct gcggctgtcg gctggtgctt ggcggcaggg acgatgtgag tgcggggctg
     4801 agaggcagcc atggggcccg cggtgagccc ttggacccgg cgcgcccctt gcagaggcct
     4861 cccagacccg aggtgcccag ggcattccgg aggcagccga gggcagcagc tcccagtttc
     4921 ttcttttcga gtattaaagg tggaagaagg tccatatctt tttctgtggg tgcttcaagt
     4981 gttgttggaa gtggaggcag cagtgacaag gggaagcttt ccctgcagga tgtagctgag
     5041 ctgattcggg ccagagcctg ccagagggtg gtggtcatgg tgggggccgg catcagcaca
     5101 cccagtggca ttccagactt cagatcgccg gggagtggcc tgtacagcaa cctccagcag
     5161 tacgatctcc cgtaccccga ggccattttt gaactcccat tcttctttca caaccccaag
     5221 ccctttttca ctttggccaa ggagctgtac cctggaaact acaagcccaa cgtcactcac
     5281 tactttctcc ggctgcttca tgacaagggg ctgcttctgc ggctctacac gcagaacatc
     5341 gatgggcttg agagagtgtc gggcatccct gcctcaaagc tggttgaagc tcatggaacc
     5401 tttgcctctg ccacctgcac agtctgccaa agacccttcc caggggagga cattcgggct
     5461 gacgtgatgg cagacagggt tccccgctgc ccggtctgca ccggcgttgt gaagcccgac
     5521 attgtgttct ttggggagcc gctgccccag aggttcttgc tgcatgtggt tgatttcccc
     5581 atggcagatc tgctgctcat ccttgggacc tccctggagg tggagccttt tgccagcttg
     5641 accgaggccg tgcggagctc agttccccga ctgctcatca accgggactt ggtggggccc
     5701 ttggcttggc atcctcgcag cagggacgtg gcccagctgg gggacgtggt tcacggcgtg
     5761 gaaagcctag tggagcttct gggctggaca gaagagatgc gggaccttgt gcagcgggaa
     5821 actgggaagc ttgatggacc agacaaagaa ttcggaggag actacaagga cgacgatgac
     5881 aagtaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     5941 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     6001 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     6061 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     6121 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     6181 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     6241 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     6301 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     6361 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     6421 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     6481 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     6541 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     6601 taacaaaaat ttaacgcg
//
