LOCUS       sequence-340128-        6434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 2xFLAG-2xSTREP-tagged cyclin D1(T286I) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6434)
  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 6434)
  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: pcDNA3.1-2xFLAG-2xSTREP-CCND1(T286I)
FEATURES             Location/Qualifiers
     source          1..6434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(80..100)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        85..414
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      265..400
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     327..346
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             481..1275
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(535..554)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1145..1164
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1449..1570
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1486..1505)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1540..1559
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1619..1635)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1619..1635)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1632..1654)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1643..1659
                     /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(1667..1673,1674..1691,1692..1697))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1712..1733
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1850..1867)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2021..2609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2101..2120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2780..3571,3572..3640))
                     /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     3403..3422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3641..3745)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3820..3839)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4011..4390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4391..4594
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4545..4565
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4639..4658
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4639..4657
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4699..4722
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4738..4761
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4777..4800
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             4837..4860
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     regulatory      5477..5486
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5801..5818)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5807..6031
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6077..6434,1..71)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6164..6183)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6374..6395
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
       61 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
      121 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
      181 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
      241 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
      301 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
      361 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
      421 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
      481 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
      541 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
      601 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
      661 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
      721 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
      781 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
      841 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
      901 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
      961 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     1021 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     1081 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     1141 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     1201 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     1261 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     1321 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     1381 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     1441 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1501 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1561 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     1621 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1681 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1741 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1801 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1861 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1921 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1981 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     2041 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     2101 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     2161 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     2221 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     2281 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     2341 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2401 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2461 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2521 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2581 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     2641 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     2701 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2761 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2821 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2881 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2941 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     3001 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     3061 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     3121 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     3181 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     3241 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     3301 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3361 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3421 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3481 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3541 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3601 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3661 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3721 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     3781 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     3841 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     3901 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     3961 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
     4021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     4081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     4141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     4201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     4261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     4321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     4381 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     4441 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     4501 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     4561 gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc
     4621 ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt
     4681 aaacttaagc ttaccatgga ctacaaggac gacgatgaca aggtaccagg atctggggat
     4741 tataaagatg atgatgataa agggtcggcc gccagctgga gccaccctca gttcgagaag
     4801 ggaggaggaa gcggcggagg cagcggagga ggaagctgga gccacccgca gttcgagaaa
     4861 ggagctggat ccagcagcga acaccagctc ctgtgctgcg aagtggaaac catccgccgc
     4921 gcgtaccccg atgccaacct cctcaacgac cgggtgctgc gggccatgct gaaggcggag
     4981 gagacctgcg cgccctcggt gtcctacttc aaatgtgtgc agaaggaggt cctgccgtcc
     5041 atgcggaaga tcgtcgccac ctggatgctg gaggtctgcg aggaacagaa gtgcgaggag
     5101 gaggtcttcc cgctggccat gaactacctg gaccgcttcc tgtcgctgga gcccgtgaaa
     5161 aagagccgcc tgcagctgct gggggccact tgcatgttcg tggcctctaa gatgaaggag
     5221 accatccccc tgacggccga gaagctgtgc atctacaccg acaactccat ccggcccgag
     5281 gagctgctgc aaatggagct gctcctggtg aacaagctca agtggaacct ggccgcaatg
     5341 accccgcacg atttcattga acacttcctc tccaaaatgc cagaggcgga ggagaacaaa
     5401 cagatcatcc gcaaacacgc gcagaccttc gttgccctct gtgccacaga tgtgaagttc
     5461 atttccaatc cgccctccat ggtggcagcg gggagcgtgg tggccgcagt gcaaggcctg
     5521 aacctgagga gccccaacaa cttcctgtcc tactaccgcc tcacacgctt cctctccaga
     5581 gtgatcaagt gtgacccaga ctgcctccgg gcctgccagg agcagatcga agccctgctg
     5641 gagtcaagcc tgcgccaggc ccagcagaac atggacccca aggccgccga ggaggaggaa
     5701 gaggaggagg aggaggtgga cctggcttgc atacccaccg acgtgcggga cgtggacatc
     5761 tgactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     5821 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     5881 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     5941 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     6001 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     6061 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6121 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6181 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6241 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6301 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6361 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6421 tattcttttg attt
//
