LOCUS       sequence-340134-        6434 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 2xFLAG-2xSTREP-tagged cyclin D1(T286A) 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(T286A)
FEATURES             Location/Qualifiers
     source          1..6434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             923..946
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             962..985
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1001..1024
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1061..1084
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     regulatory      1701..1710
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2025..2042)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2031..2255
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2301..2729
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2388..2407)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2598..2619
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2738..2758)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2743..3072
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2923..3058
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2985..3004
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3139..3933
                     /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(3193..3212)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3803..3822
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4107..4228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4144..4163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4198..4217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4277..4293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4277..4293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4290..4312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4301..4317
                     /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(4325..4331,4332..4349,4350..4355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4370..4391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4508..4525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4679..5267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4759..4778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5438..6229,6230..6298))
                     /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     6061..6080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6299..6403)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttacca tggactacaa ggacgacgat gacaaggtac caggatctgg
      961 ggattataaa gatgatgatg ataaagggtc ggccgccagc tggagccacc ctcagttcga
     1021 gaagggagga ggaagcggcg gaggcagcgg aggaggaagc tggagccacc cgcagttcga
     1081 gaaaggagct ggatccagca gcgaacacca gctcctgtgc tgcgaagtgg aaaccatccg
     1141 ccgcgcgtac cccgatgcca acctcctcaa cgaccgggtg ctgcgggcca tgctgaaggc
     1201 ggaggagacc tgcgcgccct cggtgtccta cttcaaatgt gtgcagaagg aggtcctgcc
     1261 gtccatgcgg aagatcgtcg ccacctggat gctggaggtc tgcgaggaac agaagtgcga
     1321 ggaggaggtc ttcccgctgg ccatgaacta cctggaccgc ttcctgtcgc tggagcccgt
     1381 gaaaaagagc cgcctgcagc tgctgggggc cacttgcatg ttcgtggcct ctaagatgaa
     1441 ggagaccatc cccctgacgg ccgagaagct gtgcatctac accgacaact ccatccggcc
     1501 cgaggagctg ctgcaaatgg agctgctcct ggtgaacaag ctcaagtgga acctggccgc
     1561 aatgaccccg cacgatttca ttgaacactt cctctccaaa atgccagagg cggaggagaa
     1621 caaacagatc atccgcaaac acgcgcagac cttcgttgcc ctctgtgcca cagatgtgaa
     1681 gttcatttcc aatccgccct ccatggtggc agcggggagc gtggtggccg cagtgcaagg
     1741 cctgaacctg aggagcccca acaacttcct gtcctactac cgcctcacac gcttcctctc
     1801 cagagtgatc aagtgtgacc cagactgcct ccgggcctgc caggagcaga tcgaagccct
     1861 gctggagtca agcctgcgcc aggcccagca gaacatggac cccaaggccg ccgaggagga
     1921 ggaagaggag gaggaggagg tggacctggc ttgcgcaccc accgacgtgc gggacgtgga
     1981 catctgactc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
     2041 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2101 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2161 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2221 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2281 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2341 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2401 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2461 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2521 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     2581 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     2641 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     2701 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     2761 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2821 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2881 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2941 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3001 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3061 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3121 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3181 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3241 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3301 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3361 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3421 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3481 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3541 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     3601 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     3661 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     3721 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     3781 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     3841 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     3901 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     3961 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4021 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4081 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4141 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4201 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4261 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4321 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4381 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4441 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4501 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4561 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4621 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4681 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4741 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4801 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4861 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4921 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4981 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5041 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5101 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5161 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5221 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5281 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5341 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5401 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5461 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5521 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5581 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5641 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5701 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5761 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5821 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5881 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5941 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6001 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6061 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6121 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6181 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6241 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6301 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6361 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6421 tgccacctga cgtc
//
