LOCUS       sequence-340196-        7272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for the constitutive expression of 
            FLAG-2xSTREP-tagged cyclin D2(P281L) and a puromycin resistance 
            marker in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7272)
  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 7272)
  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-CCND2(P281L)-Puro
FEATURES             Location/Qualifiers
     source          1..7272
                     /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        2446..2945
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2481..2504)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2878..2897
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2966..3565
                     /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(2966..2985)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3462..3482
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3665..4179
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4341..4357
                     /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(4365..4371,4372..4389,4390..4395))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4410..4431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4548..4565)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4719..5307)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4799..4818)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5478..6269,6270..6338))
                     /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     6101..6120
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6339..6443)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6511..6529
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6567..6589)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6689..6708
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6902..6924
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7052..7071
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7271..7272,1..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 agatctagca
     1561 gcgagctgct gtgccacgag gtggacccgg tccgcagggc cgtgcgggac cgcaacctgc
     1621 tccgagacga ccgcgtcctg cagaacctgc tcaccatcga ggagcgctac cttccgcagt
     1681 gctcctactt caagtgcgtg cagaaggaca tccaacccta catgcgcaga atggtggcca
     1741 cctggatgct ggaggtctgt gaggaacaga agtgcgaaga agaggtcttc cctctggcca
     1801 tgaattacct ggaccgtttc ttggctgggg tcccgactcc gaagtcccat ctgcaactcc
     1861 tgggtgctgt ctgcatgttc ctggcctcca aactcaaaga gaccagcccg ctgaccgcgg
     1921 agaagctgtg catttacacc gacaactcca tcaagcctca ggagctgctg gagtgggaac
     1981 tggtggtgct ggggaagttg aagtggaacc tggcagctgt cactcctcat gacttcattg
     2041 agcacatctt gcgcaagctg ccccagcagc gggagaagct gtctctgatc cgcaagcatg
     2101 ctcagacctt cattgctctg tgtgccacgg actttaagtt tgccatgtac ccaccgtcga
     2161 tgatcgcaac tggaagtgtg ggagcagcca tctgtgggct ccagcaggat gaggaagtga
     2221 gctcgctcac ttgtgatgcc ctgactgagc tgctggctaa gatcaccaac acagacgtgg
     2281 attgtctcaa agcttgccag gagcagattg aggcggtgct cctcaatagc ctgcagcagt
     2341 accgtcagga ccaacgtgac ggatccaagt cggaggatga actggaccaa gccagcaccc
     2401 ttacagacgt gcgggatatc gacctgtgac tcgaggaatt ctaccgggta ggggaggcgc
     2461 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     2521 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     2581 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     2641 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     2701 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     2761 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     2821 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     2881 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     2941 tttcgacctg cagcccaagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     3001 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3061 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3121 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3181 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3241 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3301 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     3361 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3421 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3481 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3541 catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     3601 gagcgcacga ccccatgcat cgataaaata aaagatttta tttagtctcc agaaaaaggg
     3661 gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa
     3721 ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg aacagagaga
     3781 cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     3841 caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga
     3901 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     3961 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     4021 aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtgtatc
     4081 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     4141 ctctgagtga ttgactaccc gtcagcgggg gtctttcatg ggtaacagtt tcttgaagtt
     4201 ggagaacaac attctgaggg taggagtcga atattaagta atcctgactc aattagccac
     4261 tgttttgaat ccacatactc caatactcct gaaatagttc attatggaca gcgcagaaag
     4321 agctggggag aattgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4381 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4441 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4501 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4561 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4621 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4681 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4741 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4801 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4861 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4921 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4981 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5041 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5101 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5161 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5221 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5281 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5341 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5401 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5461 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5521 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5581 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5641 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5701 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5761 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5821 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5881 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5941 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6001 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6061 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6121 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6181 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6241 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6301 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6361 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6421 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6481 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     6541 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     6601 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6661 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     6721 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat
     6781 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     6841 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     6901 tcccagtcac gacgttgtaa aacgacggcg caaggaatgg tgcatgcaag gagatggcgc
     6961 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     7021 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     7081 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg cgattagtcc
     7141 aatttgttaa agacaggata tcagtggtcc aggctctagt tttgactcaa caatatcacc
     7201 agctgaagcc tatagagtac gagccataga taaaataaaa gattttattt agtctccaga
     7261 aaaagggggg aa
//
