LOCUS       sequence-340106-        6521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 2xFLAG-2xSTREP-tagged CDK6 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6521)
  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 6521)
  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-CDK6
FEATURES             Location/Qualifiers
     source          1..6521
                     /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"
     primer_bind     complement(2112..2129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2118..2342
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2388..2816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2475..2494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2685..2706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2825..2845)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2830..3159
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3010..3145
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3072..3091
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3226..4020
                     /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(3280..3299)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3890..3909
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4194..4315
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4231..4250)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4285..4304
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4364..4380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4364..4380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4377..4399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4388..4404
                     /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(4412..4418,4419..4436,4437..4442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4457..4478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4595..4612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4766..5354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4846..4865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5525..6316,6317..6385))
                     /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     6148..6167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6386..6490)
                     /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 ggatccgaga aggacggcct gtgccgcgct gaccagcagt acgaatgcgt
     1141 ggcggagatc ggggagggcg cctatgggaa ggtgttcaag gcccgcgact tgaagaacgg
     1201 aggccgtttc gtggcgttga agcgcgtgcg ggtgcagacc ggcgaggagg gcatgccgct
     1261 ctccaccatc cgcgaggtgg cggtgctgag gcacctggag accttcgagc accccaacgt
     1321 ggtcaggttg tttgatgtgt gcacagtgtc acgaacagac agagaaacca aactaacttt
     1381 agtgtttgaa catgtcgatc aagacttgac cacttacttg gataaagttc cagagcctgg
     1441 agtgcccact gaaaccataa aggatatgat gtttcagctt ctccgaggtc tggactttct
     1501 tcattcacac cgagtagtgc atcgcgatct aaaaccacag aacattctgg tgaccagcag
     1561 cggacaaata aaactcgctg acttcggcct tgcccgcatc tatagtttcc agatggctct
     1621 aacctcagtg gtcgtcacgc tgtggtacag agcacccgaa gtcttgctcc agtccagcta
     1681 cgccaccccc gtggatctct ggagtgttgg ctgcatattt gcagaaatgt ttcgtagaaa
     1741 gcctcttttt cgtggaagtt cagatgttga tcaactagga aaaatcttgg acgtgattgg
     1801 actcccagga gaagaagact ggcctagaga tgttgccctt cccaggcagg cttttcattc
     1861 aaaatctgcc caaccaattg agaagtttgt aacagatatc gatgaactag gcaaagacct
     1921 acttctgaag tgtttgacat ttaacccagc caaaagaata tctgcctaca gtgccctgtc
     1981 tcacccatac ttccaggacc tggaaaggtg caaagaaaac ctggattccc acctgccgcc
     2041 cagccagaac acctcggagc tgaatacagc ctgactcgag tctagagggc ccgtttaaac
     2101 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2161 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2221 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2281 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2341 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2401 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2461 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2521 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2581 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2641 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2701 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2761 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     2821 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     2881 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     2941 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3001 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3061 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3121 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3181 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3241 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3301 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3361 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3421 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3481 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3541 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3601 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3661 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3721 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     3781 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3841 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3901 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     3961 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4021 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4081 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4141 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4201 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4261 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4321 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4381 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4441 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4561 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4621 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4681 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4741 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4801 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4861 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4921 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4981 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5041 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5101 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5161 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5221 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5281 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5341 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5401 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5461 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5521 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5581 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5641 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5701 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5761 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5821 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5881 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5941 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6001 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6061 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6121 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6181 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6241 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6301 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6361 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6421 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6481 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
