LOCUS       sequence-151245-        6349 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of human SIRT4 with flag tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6349)
  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 6349)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: SIRT4 Flag
FEATURES             Location/Qualifiers
     source          1..6349
                     /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"
     CDS             1882..1905
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1940..1957)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1946..2170
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2216..2644
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2303..2322)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2513..2534
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2653..2673)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2658..2987
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2838..2973
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2900..2919
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3054..3848
                     /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(3108..3127)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3718..3737
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4022..4143
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4059..4078)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4113..4132
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4192..4208)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4192..4208)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4205..4227)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4216..4232
                     /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(4240..4246,4247..4264,4265..4270))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4285..4306
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4423..4440)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4594..5182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4674..4693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5353..6144,6145..6213))
                     /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     5976..5995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6214..6318)
                     /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 gctggctagc
      901 gtttaaactt aagcttgcgg ccgcatgaag atgagctttg cgttgacttt caggtcagca
      961 aaaggccgtt ggatcgcaaa ccccagccag ccgtgctcga aagcctccat tgggttattt
     1021 gtgccagcaa gtcctcctct ggaccctgag aaggtcaaag agttacagcg cttcatcacc
     1081 ctttccaaga gactccttgt gatgactggg gcaggaatct ccaccgaatc ggggatacca
     1141 gactacaggt cagaaaaagt ggggctttat gcccgcactg accgcaggcc catccagcat
     1201 ggtgattttg tccggagtgc cccaatccgc cagcggtact gggcgagaaa cttcgtaggc
     1261 tggcctcaat tctcctccca ccagcctaac cctgcacact gggctttgag cacctgggag
     1321 aaactcggaa agctgtactg gttggtgacc caaaatgtgg atgctttgca caccaaggcg
     1381 gggagtcggc gcctgacaga gctccacgga tgcatggaca gggtcctgtg cttggattgt
     1441 ggggaacaga ctccccgggg ggtgctgcaa gagcgtttcc aagtcctgaa ccccacctgg
     1501 agtgctgagg cccatggcct ggctcctgat ggtgacgtct ttctctcaga ggagcaagtc
     1561 cggagctttc aggtcccaac ctgcgttcaa tgtggaggcc atctgaaacc agatgtcgtt
     1621 ttcttcgggg acacagtgaa ccctgacaag gttgattttg tgcacaagcg tgtaaaagaa
     1681 gccgactccc tcttggtggt gggatcatcc ttgcaggtat actctggtta caggtttatc
     1741 ctcactgcct gggagaagaa gctcccgatt gcaatactga acattgggcc cacacggtcg
     1801 gatgacttgg cgtgtctgaa actgaattct cgttgtggag agttgctgcc tttgatagac
     1861 ccatgcagcg gccgcggagg agactacaag gacgacgatg acaagtaatc tagagggccc
     1921 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1981 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2041 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2101 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     2161 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2221 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2281 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2341 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2401 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2461 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2521 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2581 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2641 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2701 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2761 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2821 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2881 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     2941 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3001 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3061 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3121 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3181 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3241 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3301 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3361 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3421 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3481 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3541 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3601 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3661 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3721 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3781 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3841 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     3901 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     3961 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4021 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4081 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4141 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4201 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4261 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4321 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4381 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4441 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4501 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4561 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4621 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4681 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4741 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4801 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4861 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4921 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4981 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5041 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5101 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5161 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5221 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5281 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5341 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5401 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5461 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5521 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5581 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5641 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5701 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5761 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5821 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5881 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5941 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6001 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6061 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6121 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6181 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6241 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6301 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
