LOCUS       sequence-340122-        6484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 2xFLAG-2xSTREP-tagged CDK2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6484)
  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 6484)
  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-CDK2
FEATURES             Location/Qualifiers
     source          1..6484
                     /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(2075..2092)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2081..2305
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2351..2779
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2438..2457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2648..2669
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2788..2808)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2793..3122
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2973..3108
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3035..3054
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3189..3983
                     /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(3243..3262)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3853..3872
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4157..4278
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4194..4213)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4248..4267
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4327..4343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4327..4343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4340..4362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4351..4367
                     /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(4375..4381,4382..4399,4400..4405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4420..4441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4558..4575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4729..5317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4809..4828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5488..6279,6280..6348))
                     /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     6111..6130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6349..6453)
                     /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 ggatccagtg tggtggaatt ggagaacttc caaaaggtgg aaaagatcgg
     1141 agagggcacg tacggagttg tgtacaaagc cagaaacaag ttgacgggag aggtggtggc
     1201 gcttaagaaa atccgcctgg acactgagac tgagggtgtg cccagtactg ccatccgaga
     1261 gatctctctg cttaaggagc ttaaccatcc taatattgtc aagctgctgg atgtcattca
     1321 cacagaaaat aaactctacc tggtttttga atttctgcac caagatctca agaaattcat
     1381 ggatgcctct gctctcactg gcattcctct tcccctcatc aagagctatc tgttccagct
     1441 gctccagggc ctagctttct gccattctca tcgggtcctc caccgagacc ttaaacctca
     1501 gaatctgctt attaacacag agggggccat caagctagca gactttggac tagccagagc
     1561 ttttggagtc cctgttcgta cttacaccca tgaggtggtg accctgtggt accgagctcc
     1621 tgaaatcctc ctgggctgca aatattattc cacagctgtg gacatctgga gcctgggctg
     1681 catctttgct gagatggtga ctcgccgggc cctattccct ggagattctg agattgacca
     1741 gctcttccgg atctttcgga ctctggggac cccagatgag gtggtgtggc caggagttac
     1801 ttctatgcct gattacaagc caagtttccc caagtgggcc cggcaagatt ttagtaaagt
     1861 tgtacctccc ctggatgaag atggacggag cttgttatcg caaatgctgc actacgaccc
     1921 taacaagcgg atttcggcca aggcagccct ggctcaccct ttcttccagg atgtgaccaa
     1981 gccagtaccc catcttcgac tctgaaattc tgcagatatc cagcacagtg gcggccgctc
     2041 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2101 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2161 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2221 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2281 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2341 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2401 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2461 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2521 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2581 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     2641 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     2701 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     2761 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2821 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2881 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2941 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3001 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3061 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3121 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3181 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3241 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     3301 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     3361 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     3421 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     3481 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     3541 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     3601 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     3661 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     3721 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     3781 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     3841 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     3901 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     3961 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4021 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4081 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4141 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4201 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4261 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4321 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4381 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4441 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4501 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4561 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4621 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4681 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4741 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4801 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4861 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4921 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4981 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5041 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5101 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5161 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5221 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5281 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5341 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5401 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5461 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5521 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5581 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5641 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5701 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5761 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5821 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5881 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5941 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6001 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6061 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6121 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6181 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6241 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6301 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6361 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6421 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6481 cgtc
//
