LOCUS       sequence-340125-        6488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 2xFLAG-2xSTREP-tagged CDK4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6488)
  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 6488)
  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-CDK4
FEATURES             Location/Qualifiers
     source          1..6488
                     /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(2079..2096)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2085..2309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2355..2783
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2442..2461)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2652..2673
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2792..2812)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2797..3126
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2977..3112
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3039..3058
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3193..3987
                     /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(3247..3266)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3857..3876
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4161..4282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4198..4217)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4252..4271
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4331..4347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4331..4347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4344..4366)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4355..4371
                     /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(4379..4385,4386..4403,4404..4409))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4424..4445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4562..4579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4733..5321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4813..4832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5492..6283,6284..6352))
                     /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     6115..6134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6353..6457)
                     /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 caggatctat
      961 ggattataaa gatgatgatg ataaagggtc ggccgccagc tggagccacc ctcagttcga
     1021 gaagggagga ggaagcggcg gaggcagcgg aggaggaagc tggagccacc cgcagttcga
     1081 gaaaggagct ggatccatgg ctacctctcg atatgagcca gtggctgaaa ttggtgtcgg
     1141 tgcctatggg acagtgtaca aggcccgtga tccccacagt ggccactttg tggccctcaa
     1201 gagtgtgaga gtccccaatg gaggaggagg tggaggaggc cttcccatca gcacagttcg
     1261 tgaggtggct ttactgaggc gactggaggc ttttgagcat cccaatgttg tccggctgat
     1321 ggacgtctgt gccacatccc gaactgaccg ggagatcaag gtaaccctgg tgtttgagca
     1381 tgtagaccag gacctaagga catatctgga caaggcaccc ccaccaggct tgccagccga
     1441 aacgatcaag gatctgatgc gccagtttct aagaggccta gatttccttc atgccaattg
     1501 catcgttcac cgagatctga agccagagaa cattctggtg acaagtggtg gaacagtcaa
     1561 gctggctgac tttggcctgg ccagaatcta cagctaccag atggcactta cacccgtggt
     1621 tgttacactc tggtaccgag ctcccgaagt tcttctgcag tccacatatg caacacctgt
     1681 ggacatgtgg agtgttggct gtatctttgc agagatgttt cgtcgaaagc ctctcttctg
     1741 tggaaactct gaagccgacc agttgggcaa aatctttgac ctgattgggc tgcctccaga
     1801 ggatgactgg cctcgagatg tatccctgcc ccgtggagcc tttcccccca gagggccccg
     1861 cccagtgcag tcggtggtac ctgagatgga ggagtcggga gcacagctgc tgctggaaat
     1921 gctgactttt aacccacaca agcgaatctc tgcctttcga gctctgcagc actcttatct
     1981 acataaggat gaaggtaatc cggagtgaga attctgcaga tatccagcac agtggcggcc
     2041 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2101 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2161 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2221 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2281 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2341 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2401 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2461 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2521 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2581 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2641 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2701 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2761 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     2821 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2881 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2941 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3001 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3061 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3121 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga
     3181 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     3241 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     3301 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     3361 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     3421 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     3481 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     3541 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     3601 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3661 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3721 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3781 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3841 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3901 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3961 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     4021 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     4081 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     4141 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
     4201 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4261 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
     4321 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4381 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4441 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4501 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4561 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4621 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4681 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4741 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4801 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4861 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4921 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4981 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5041 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5101 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5161 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5221 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5281 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5341 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5401 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5461 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5521 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5581 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5641 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5701 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5761 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5821 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5881 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5941 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6001 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6061 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6121 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6181 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6241 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6301 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6361 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6421 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6481 ctgacgtc
//
