LOCUS       sequence-340204-        7290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for the constitutive expression of 
            FLAG-2xSTREP-tagged cyclin D1(T286A) and a puromycin resistance 
            marker in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7290)
  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 7290)
  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-CCND1(T286A)-Puro
FEATURES             Location/Qualifiers
     source          1..7290
                     /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"
     regulatory      2161..2170
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        2464..2963
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2499..2522)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2896..2915
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2984..3583
                     /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(2984..3003)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3480..3500
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3683..4197
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4359..4375
                     /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(4383..4389,4390..4407,4408..4413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4428..4449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4566..4583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4737..5325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4817..4836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5496..6287,6288..6356))
                     /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     6119..6138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6357..6461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6529..6547
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6585..6607)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6707..6726
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6920..6942
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7070..7089
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7289..7290,1..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 gcgaacacca gctcctgtgc tgcgaagtgg aaaccatccg ccgcgcgtac cccgatgcca
     1621 acctcctcaa cgaccgggtg ctgcgggcca tgctgaaggc ggaggagacc tgcgcgccct
     1681 cggtgtccta cttcaaatgt gtgcagaagg aggtcctgcc gtccatgcgg aagatcgtcg
     1741 ccacctggat gctggaggtc tgcgaggaac agaagtgcga ggaggaggtc ttcccgctgg
     1801 ccatgaacta cctggaccgc ttcctgtcgc tggagcccgt gaaaaagagc cgcctgcagc
     1861 tgctgggggc cacttgcatg ttcgtggcct ctaagatgaa ggagaccatc cccctgacgg
     1921 ccgagaagct gtgcatctac accgacaact ccatccggcc cgaggagctg ctgcaaatgg
     1981 agctgctcct ggtgaacaag ctcaagtgga acctggccgc aatgaccccg cacgatttca
     2041 ttgaacactt cctctccaaa atgccagagg cggaggagaa caaacagatc atccgcaaac
     2101 acgcgcagac cttcgttgcc ctctgtgcca cagatgtgaa gttcatttcc aatccgccct
     2161 ccatggtggc agcggggagc gtggtggccg cagtgcaagg cctgaacctg aggagcccca
     2221 acaacttcct gtcctactac cgcctcacac gcttcctctc cagagtgatc aagtgtgacc
     2281 cagactgcct ccgggcctgc caggagcaga tcgaagccct gctggagtca agcctgcgcc
     2341 aggcccagca gaacatggac cccaaggccg ccgaggagga ggaagaggag gaggaggagg
     2401 tggacctggc ttgcgcaccc accgacgtgc gggacgtgga catctgactc gaggaattct
     2461 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc
     2521 tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta
     2581 ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag
     2641 tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag
     2701 cacgtctcac tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc
     2761 tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa
     2821 ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt
     2881 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct
     2941 cttcctcatc tccgggcctt tcgacctgca gcccaagctt accatgaccg agtacaagcc
     3001 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     3061 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     3121 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     3181 cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3241 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3301 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3361 caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3421 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3481 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3541 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc
     3601 gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg ataaaataaa agattttatt
     3661 tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta
     3721 agtaacgcca ttttgcaagg catggaaaat acataactga gaatagagaa gttcagatca
     3781 aggttaggaa cagagagaca gcagaatatg ggccaaacag gatatctgtg gtaagcagtt
     3841 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc cctcagcagt
     3901 ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     3961 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     4021 ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata gactgcgtcg
     4081 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     4141 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatggg
     4201 taacagtttc ttgaagttgg agaacaacat tctgagggta ggagtcgaat attaagtaat
     4261 cctgactcaa ttagccactg ttttgaatcc acatactcca atactcctga aatagttcat
     4321 tatggacagc gcagaaagag ctggggagaa ttgtgaaatt gttatccgct cacaattcca
     4381 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4441 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4501 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4561 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4621 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4681 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4741 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4801 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4861 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4921 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4981 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5041 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5101 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5161 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5221 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5281 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5341 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5401 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5461 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5521 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5581 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5641 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5701 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5761 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5821 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5881 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5941 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6001 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6061 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6121 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6181 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6241 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6301 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6361 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6421 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6481 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6541 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     6601 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     6661 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     6721 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     6781 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     6841 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     6901 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggcgca aggaatggtg
     6961 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     7021 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     7081 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     7141 cgtagaggcg attagtccaa tttgttaaag acaggatatc agtggtccag gctctagttt
     7201 tgactcaaca atatcaccag ctgaagccta tagagtacga gccatagata aaataaaaga
     7261 ttttatttag tctccagaaa aaggggggaa
//
