LOCUS       sequence-340124-        6359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HA-tagged CDK4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6359)
  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 6359)
  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-HA-CDK4
FEATURES             Location/Qualifiers
     source          1..6359
                     /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             926..952
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1950..1967)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1956..2180
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2226..2654
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2313..2332)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2523..2544
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2663..2683)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2668..2997
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2848..2983
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2910..2929
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3064..3858
                     /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(3118..3137)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3728..3747
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4032..4153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4069..4088)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4123..4142
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4202..4218)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4202..4218)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4215..4237)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4226..4242
                     /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(4250..4256,4257..4274,4275..4280))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4295..4316
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4433..4450)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4604..5192)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4684..4703)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5363..6154,6155..6223))
                     /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     5986..6005
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6224..6328)
                     /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 tgggataccc atacgatgtt ccagattacg ctgtggtacc
      961 aggatccatg gctacctctc gatatgagcc agtggctgaa attggtgtcg gtgcctatgg
     1021 gacagtgtac aaggcccgtg atccccacag tggccacttt gtggccctca agagtgtgag
     1081 agtccccaat ggaggaggag gtggaggagg ccttcccatc agcacagttc gtgaggtggc
     1141 tttactgagg cgactggagg cttttgagca tcccaatgtt gtccggctga tggacgtctg
     1201 tgccacatcc cgaactgacc gggagatcaa ggtaaccctg gtgtttgagc atgtagacca
     1261 ggacctaagg acatatctgg acaaggcacc cccaccaggc ttgccagccg aaacgatcaa
     1321 ggatctgatg cgccagtttc taagaggcct agatttcctt catgccaatt gcatcgttca
     1381 ccgagatctg aagccagaga acattctggt gacaagtggt ggaacagtca agctggctga
     1441 ctttggcctg gccagaatct acagctacca gatggcactt acacccgtgg ttgttacact
     1501 ctggtaccga gctcccgaag ttcttctgca gtccacatat gcaacacctg tggacatgtg
     1561 gagtgttggc tgtatctttg cagagatgtt tcgtcgaaag cctctcttct gtggaaactc
     1621 tgaagccgac cagttgggca aaatctttga cctgattggg ctgcctccag aggatgactg
     1681 gcctcgagat gtatccctgc cccgtggagc ctttcccccc agagggcccc gcccagtgca
     1741 gtcggtggta cctgagatgg aggagtcggg agcacagctg ctgctggaaa tgctgacttt
     1801 taacccacac aagcgaatct ctgcctttcg agctctgcag cactcttatc tacataagga
     1861 tgaaggtaat ccggagtgag aattctgcag atatccagca cagtggcggc cgctcgagtc
     1921 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1981 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2041 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2101 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2161 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     2221 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2281 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2341 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     2401 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     2461 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2521 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2581 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2641 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2701 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2761 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2821 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2881 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2941 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3001 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     3061 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3121 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3181 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3241 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3301 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3361 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3421 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3481 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3541 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     3601 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3661 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3721 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3781 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3841 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     3901 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3961 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4021 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4081 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4141 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     4201 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4261 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5041 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5281 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5341 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5401 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5461 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5521 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5581 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5641 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5701 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5761 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5821 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5881 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5941 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     6001 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6061 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6121 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6181 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6241 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6301 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
