LOCUS       sequence-344922-        7260 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Retroviral vector for the constitutive expression of 
            FLAG-2xSTREP-tagged cyclin D2 and a puromycin resistance marker in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7260)
  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 7260)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Puro
FEATURES             Location/Qualifiers
     source          1..7260
                     /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..2933
                     /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     2866..2885
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2954..3553
                     /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(2954..2973)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3450..3470
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3653..4167
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4329..4345
                     /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(4353..4359,4360..4377,4378..4383))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4398..4419
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4536..4553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4707..5295)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4787..4806)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5466..6257,6258..6326))
                     /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     6089..6108
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6327..6431)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6499..6517
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6555..6577)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6677..6696
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6890..6912
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7040..7059
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7259..7260,1..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 ctacagacgt 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 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc
     2881 aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca
     2941 gcccaagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3001 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     3061 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     3121 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     3181 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     3241 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     3301 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     3361 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     3421 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     3481 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     3541 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     3601 ccatgcatcg ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     3661 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat
     3721 acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg
     3781 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     3841 gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     3901 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     3961 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     4021 ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc
     4081 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt
     4141 gactacccgt cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat
     4201 tctgagggta ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc
     4261 acatactcca atactcctga aatagttcat tatggacagc gcagaaagag ctggggagaa
     4321 ttgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4381 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4441 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4501 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4561 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4621 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4681 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4741 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4801 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4861 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4921 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4981 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5041 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5101 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5161 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5221 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5281 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5341 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5401 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5461 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5521 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5581 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5641 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5701 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5761 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5821 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5881 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5941 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6001 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6061 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6121 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6181 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6241 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6301 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6361 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6421 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6481 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     6541 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     6601 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     6661 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     6721 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
     6781 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     6841 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     6901 cgttgtaaaa cgacggcgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6961 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     7021 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     7081 cgccggtgat gccggccacg atgcgtccgg cgtagaggcg attagtccaa tttgttaaag
     7141 acaggatatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta
     7201 tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa
//
