LOCUS       sequence-340206-        7281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for the constitutive expression of 
            FLAG-2xSTREP-tagged cyclin D3(T283A) and a puromycin resistance 
            marker in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7281)
  AUTHORS   Simoneschi D, Rona G, Zhou N, Jeong YT, Jiang S, Milletti G, Arbini 
            AA, O'Sullivan A, Wang AA, Nithikasem S, Keegan S, Siu Y, 
            Cianfanelli V, Maiani E, Nazio F, Cecconi F, Boccalatte F, Fenyo D, 
            Jones DR, Busino L, Pagano M
  TITLE     CRL4(AMBRA1) is a master regulator of D-type cyclins.
  JOURNAL   Nature. 2021 Apr;592(7856):789-793. doi: 10.1038/s41586-021-03445-y.
            Epub 2021 Apr 14.
  PUBMED    33854235
REFERENCE   2  (bases 1 to 7281)
  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"; doi: 
            "10.1038/s41586-021-03445-y"; journalName: "Nature"; date: 
            "2021-04"; volume: "592"; issue: "7856"; pages: "789-793"
COMMENT     Sequence Label: pMSCV-FLAG-2xSTREP-CCND3(T283A)-Puro
FEATURES             Location/Qualifiers
     source          1..7281
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1416..1425
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1425..1448
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1464..1487
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1524..1547
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        2455..2954
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2490..2513)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2887..2906
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2975..3574
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2975..2994)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3471..3491
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3674..4188
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4350..4366
                     /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(4374..4380,4381..4398,4399..4404))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4419..4440
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4557..4574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4728..5316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4808..4827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5487..6278,6279..6347))
                     /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     6110..6129
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6348..6452)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6520..6538
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6576..6598)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6698..6717
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6911..6933
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7061..7080
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7280..7281,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatccgccac catggattat aaagatgatg
     1441 atgataaagg gtcggccgcc agctggagcc accctcagtt cgagaaggga ggaggaagcg
     1501 gcggaggcag cggaggagga agctggagcc acccgcagtt cgagaaagga agatccagca
     1561 gcgagctgct gtgttgcgaa ggcacccggc acgcgccccg ggccgggccg gacccgcggc
     1621 tgctggggga ccagcgtgtc ctgcagagcc tgctccgcct ggaggagcgc tacgtacccc
     1681 gcgcctccta cttccagtgc gtgcagcggg agatcaagcc gcacatgcgg aagatgctgg
     1741 cttactggat gctggaggta tgtgaggagc agcgctgtga ggaggaagtc ttccccctgg
     1801 ccatgaacta cctggatcgc tacctgtctt gcgtccccac ccgaaaggcg cagttgcagc
     1861 tcctgggtgc ggtctgcatg ctgctggcct ccaagctgcg cgagaccacg cccctgacca
     1921 tcgaaaaact gtgcatctac accgaccacg ctgtctctcc ccgccagttg cgggactggg
     1981 aggtgctggt cctagggaag ctcaagtggg acctggctgc tgtgattgca catgatttcc
     2041 tggccttcat tctgcaccgg ctctctctgc cccgtgaccg acaggccttg gtcaaaaagc
     2101 atgcccagac ctttttggcc ctctgtgcta cagattatac ctttgccatg tacccgccat
     2161 ccatgatcgc cacgggcagc attggggctg cagtgcaagg cctgggtgcc tgctccatgt
     2221 ccggggatga gctcacagag ctgctggcag ggatcactgg cactgaagtg gactgcctgc
     2281 gggcctgtca ggagcagatc gaagctgcac tcagggagag cctcagggaa gccgctcaga
     2341 ccagctccag cccagcgccc aaagcccccc ggggctccag cagccaaggg cccagccaga
     2401 ccagcgctcc tacagatgtc acagccatac acctgtagct cgaggaattc taccgggtag
     2461 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     2521 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     2581 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     2641 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     2701 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     2761 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     2821 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
     2881 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     2941 ctccgggcct ttcgacctgc agcccaagct taccatgacc gagtacaagc ccacggtgcg
     3001 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     3061 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     3121 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     3181 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     3241 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     3301 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     3361 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     3421 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     3481 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     3541 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
     3601 gaccgaaagg agcgcacgac cccatgcatc gataaaataa aagattttat ttagtctcca
     3661 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc
     3721 attttgcaag gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga
     3781 acagagagac agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg
     3841 gctcagggcc aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga
     3901 accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac
     3961 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa
     4021 agagcccaca acccctcact cggcgcgcca gtcctccgat agactgcgtc gcccgggtac
     4081 ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg
     4141 gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcatgg gtaacagttt
     4201 cttgaagttg gagaacaaca ttctgagggt aggagtcgaa tattaagtaa tcctgactca
     4261 attagccact gttttgaatc cacatactcc aatactcctg aaatagttca ttatggacag
     4321 cgcagaaaga gctggggaga attgtgaaat tgttatccgc tcacaattcc acacaacata
     4381 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4441 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4501 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4561 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4621 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4681 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4741 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4801 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4861 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4921 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4981 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5041 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5101 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5161 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5221 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5281 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5341 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5401 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5461 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5521 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5581 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5641 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5701 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5761 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5821 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5881 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5941 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6001 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6061 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6121 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6181 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6241 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6301 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6361 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6421 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6481 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     6541 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     6601 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     6661 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     6721 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     6781 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     6841 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     6901 ccagggtttt cccagtcacg acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg
     6961 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     7021 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     7081 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagaggc
     7141 gattagtcca atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac
     7201 aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta
     7261 gtctccagaa aaagggggga a
//
