LOCUS       sequence-483552-        7107 bp    DNA     circular SYN 15-AUG-2025
DEFINITION  Expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7107)
  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 7107)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 15, 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-SIRT4
FEATURES             Location/Qualifiers
     source          1..7107
                     /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(1920..1944)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1955..1957,1958..1960,1961..2674)
                     /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(1988..2016)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2611..2631
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2696..2713)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2702..2926
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2972..3400
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3059..3078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3269..3290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3409..3429)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3414..3743
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3594..3729
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3656..3675
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3810..4604
                     /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(3864..3883)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4474..4493
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4780..4901
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4817..4836)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4871..4890
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4950..4966)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4950..4966)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4963..4985)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4974..4990
                     /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(4998..5004,5005..5022,5023..5028))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5043..5064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5181..5198)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5352..5940)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5432..5451)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6111..6902,6903..6971))
                     /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     6734..6753
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6972..7076)
                     /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 gaagatgagc tttgcgttga ctttcaggtc agcaaaaggc cgttggatcg caaaccccag
     1021 ccagccgtgc tcgaaagcct ccattgggtt atttgtgcca gcaagtcctc ctctggaccc
     1081 tgagaaggtc aaagagttac agcgcttcat caccctttcc aagagactcc ttgtgatgac
     1141 tggggcagga atctccaccg aatcggggat accagactac aggtcagaaa aagtggggct
     1201 ttatgcccgc actgaccgca ggcccatcca gcatggtgat tttgtccgga gtgccccaat
     1261 ccgccagcgg tactgggcga gaaacttcgt aggctggcct caattctcct cccaccagcc
     1321 taaccctgca cactgggctt tgagcacctg ggagaaactc ggaaagctgt actggttggt
     1381 gacccaaaat gtggatgctt tgcacaccaa ggcggggagt cggcgcctga cagagctcca
     1441 cggatgcatg gacagggtcc tgtgcttgga ttgtggggaa cagactcccc ggggggtgct
     1501 gcaagagcgt ttccaagtcc tgaaccccac ctggagtgct gaggcccatg gcctggctcc
     1561 tgatggtgac gtctttctct cagaggagca agtccggagc tttcaggtcc caacctgcgt
     1621 tcaatgtgga ggccatctga aaccagatgt cgttttcttc ggggacacag tgaaccctga
     1681 caaggttgat tttgtgcaca agcgtgtaaa agaagccgac tccctcttgg tggtgggatc
     1741 atccttgcag gtatactctg gttacaggtt tatcctcact gcctgggaga agaagctccc
     1801 gattgcaata ctgaacattg ggcccacacg gtcggatgac ttggcgtgtc tgaaactgaa
     1861 ttctcgttgt ggagagttgc tgcctttgat agacccatgc tttaagggtg ggcgcgccga
     1921 cccagctttc ttgtacaaag tggttcgatc tagaatggct agcaaaggag aagaactttt
     1981 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc
     2041 tgtcagtgga gagggtgaag gtgatgctac atacggaaag cttaccctta aatttatttg
     2101 cactactgga aaactacctg ttccatggcc aacacttgtc actactttct cttatggtgt
     2161 tcaatgcttt tcccgttatc cggatcatat gaaacggcat gactttttca agagtgccat
     2221 gcccgaaggt tatgtacagg aacgcactat atctttcaaa gatgacggga actacaagac
     2281 gcgtgctgaa gtcaagtttg aaggtgatac ccttgttaat cgtatcgagt taaaaggtat
     2341 tgattttaaa gaagatggaa acattctcgg acacaaactc gagtacaact ataactcaca
     2401 caatgtatac atcacggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattcg
     2461 ccacaacatt gaagatggat ccgttcaact agcagaccat tatcaacaaa atactccaat
     2521 tggcgatggc cctgtccttt taccagacaa ccattacctg tcgacacaat ctgccctttc
     2581 gaaagatccc aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg
     2641 gattacacat ggcatggatg agctctacaa ataatgaatt aaacccgctg atcagcctcg
     2701 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2761 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2821 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2881 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2941 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     3001 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3061 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3121 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3181 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3241 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3301 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3361 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     3421 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3481 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3541 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3601 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3661 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3721 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3781 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3841 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3901 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3961 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     4021 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4081 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4141 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4201 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4261 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4321 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4381 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4441 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4501 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4561 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4621 cgcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     4681 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     4741 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     4801 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4861 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     4921 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4981 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5041 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5101 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5161 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5221 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5281 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5341 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5401 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5461 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5521 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5581 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5641 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5701 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5761 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5821 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5881 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     5941 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6001 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6061 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6121 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6181 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6241 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6301 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6361 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6421 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6481 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6541 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6601 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6661 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6721 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6781 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6841 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6901 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6961 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7021 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7081 tttccccgaa aagtgccacc tgacgtc
//
