LOCUS       sequence-340199-        6987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for the constitutive expression of 
            FLAG-2xSTREP-tagged p27 and a puromycin resistance marker in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6987)
  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 6987)
  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-CDKN1B-Puro
FEATURES             Location/Qualifiers
     source          1..6987
                     /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        2161..2660
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2196..2219)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2593..2612
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2681..3280
                     /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(2681..2700)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3177..3197
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3380..3894
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4056..4072
                     /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(4080..4086,4087..4104,4105..4110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4125..4146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4263..4280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4434..5022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4514..4533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5193..5984,5985..6053))
                     /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     5816..5835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6054..6158)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6226..6244
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6282..6304)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6404..6423
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6617..6639
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6767..6786
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6986..6987,1..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 agatcttcaa
     1561 acgtgcgagt gtctaacggg agccctagcc tggagcggat ggacgccagg caggcggagc
     1621 accccaagcc ctcggcctgc aggaacctct tcggcccggt ggaccacgaa gagttaaccc
     1681 gggacttgga gaagcactgc agagacatgg aagaggcgag ccagcgcaag tggaatttcg
     1741 attttcagaa tcacaaaccc ctagagggca agtacgagtg gcaagaggtg gagaagggca
     1801 gcttgcccga gttctactac agacccccgc ggccccccaa aggtgcctgc aaggtgccgg
     1861 cgcaggagag ccaggatgtc agcgggagcc gcccggcggc gcctttaatt ggggctccgg
     1921 ctaactctga ggacacgcat ttggtggacc caaagactga tccgtcggac agccagacgg
     1981 ggttagcgga gcaatgcgca ggaataagga agcgacctgc aaccgacgat tcttctactc
     2041 aaaacaaaag agccaacaga acagaagaaa atgtttcaga cggttcccca aatgccggtt
     2101 ctgtggagca gacgcccaag aagcctggcc tcagaagacg tcaaacgtaa gaattctacc
     2161 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     2221 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     2281 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     2341 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     2401 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     2461 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     2521 gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct
     2581 ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt
     2641 cctcatctcc gggcctttcg acctgcagcc caagcttacc atgaccgagt acaagcccac
     2701 ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt
     2761 cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac
     2821 cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc
     2881 ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt
     2941 gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca
     3001 acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     3061 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     3121 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     3181 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     3241 accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca
     3301 gcgcccgacc gaaaggagcg cacgacccca tgcatcgata aaataaaaga ttttatttag
     3361 tctccagaaa aaggggggaa tgaaagaccc cacctgtagg tttggcaagc tagcttaagt
     3421 aacgccattt tgcaaggcat ggaaaataca taactgagaa tagagaagtt cagatcaagg
     3481 ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct
     3541 gccccggctc agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc
     3601 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
     3661 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
     3721 aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc
     3781 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
     3841 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcatgggtaa
     3901 cagtttcttg aagttggaga acaacattct gagggtagga gtcgaatatt aagtaatcct
     3961 gactcaatta gccactgttt tgaatccaca tactccaata ctcctgaaat agttcattat
     4021 ggacagcgca gaaagagctg gggagaattg tgaaattgtt atccgctcac aattccacac
     4081 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4141 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4201 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4261 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4321 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4381 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4441 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4501 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4561 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4621 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4681 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4741 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4801 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4861 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4921 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4981 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5041 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5101 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5161 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5221 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5281 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5341 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5401 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5461 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5521 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5581 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5641 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5701 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5761 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5821 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5881 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5941 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6001 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6061 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6121 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6181 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     6241 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     6301 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     6361 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     6421 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     6481 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     6541 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     6601 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggcgcaagg aatggtgcat
     6661 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     6721 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     6781 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     6841 agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga
     6901 ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt
     6961 tatttagtct ccagaaaaag gggggaa
//
