LOCUS       sequence-175298-        6539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express CHK1 with a FLAG tag at N-terminus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6539)
  AUTHORS   Xu Y, Anderson DE, Ye Y
  TITLE     The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble 
            proteins for degradation.
  JOURNAL   Cell Discov. 2016 Nov 8;2:16040. eCollection 2016.
  PUBMED    27867533
REFERENCE   2  (bases 1 to 6539)
  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"; journalName: "Cell 
            Discov."; date: "2016-11-8"; volume: "2"; pages: "16040. eCollection
            2016"
COMMENT     Sequence Label: pcDNA5/FRT/TO-FLAG-CHK1
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        31..410
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        411..614
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     565..585
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    616..634
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    637..655
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     665..689
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      777..786
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             786..809
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2287..2304)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2293..2517
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2650..2669)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2801..2848
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2856..3878
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2856..3878
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4008..4129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4045..4064)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4099..4118
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4178..4194)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4178..4194)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4191..4213)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4202..4218
                     /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(4226..4232,4233..4250,4251..4256))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4271..4292
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4409..4426)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4580..5168)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4660..4679)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5339..6130,6131..6199))
                     /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     5962..5981
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6200..6304)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6379..6398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
       61 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      121 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      181 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      241 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      301 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      361 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      421 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      481 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      541 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      601 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      661 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      721 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaaggccg
      781 ccatggatta caaggatgat gatgataagc ttggtaccat ggcagtgccc tttgtggaag
      841 actgggactt ggtgcaaacc ctgggagaag gtgcctatgg agaagttcaa cttgctgtga
      901 atagagtaac tgaagaagca gtcgcagtga agattgtaga tatgaagcgt gccgtagact
      961 gtccagaaaa tattaagaaa gagatctgta tcaataaaat gctaaatcat gaaaatgtag
     1021 taaaattcta tggtcacagg agagaaggca atatccaata tttatttctg gagtactgta
     1081 gtggaggaga gctttttgac agaatagagc cagacatagg catgcctgaa ccagatgctc
     1141 agagattctt ccatcaactc atggcagggg tggtttatct gcatggtatt ggaataactc
     1201 acagggatat taaaccagaa aatcttctgt tggatgaaag ggataacctc aaaatctcag
     1261 actttggctt ggcaacagta tttcggtata ataatcgtga gcgtttgttg aacaagatgt
     1321 gtggtacttt accatatgtt gctccagaac ttctgaagag aagagaattt catgcagaac
     1381 cagttgatgt ttggtcctgt ggaatagtac ttactgcaat gctcgctgga gaattgccat
     1441 gggaccaacc cagtgacagc tgtcaggagt attctgactg gaaagaaaaa aaaacatacc
     1501 tcaacccttg gaaaaaaatc gattctgctc ctctagctct gctgcataaa atcttagttg
     1561 agaatccatc agcaagaatt accattccag acatcaaaaa agatagatgg tacaacaaac
     1621 ccctcaagaa aggggcaaaa aggccccgag tcacttcagg tggtgtgtca gagtctccca
     1681 gtggattttc taagcacatt caatccaatt tggacttctc tccagtaaac agtgcttcta
     1741 gtgaagaaaa tgtgaagtac tccagttctc agccagaacc ccgcacaggt ctttccttat
     1801 gggataccag cccctcatac attgataaat tggtacaagg gatcagcttt tcccagccca
     1861 catgtcctga tcatatgctt ttgaatagtc agttacttgg caccccagga tcctcacaga
     1921 acccctggca gcggttggtc aaaagaatga cacgattctt taccaaattg gatgcagaca
     1981 aatcttatca atgcctgaaa gagacttgtg agaagttggg ctatcaatgg aagaaaagtt
     2041 gtatgaatca ggttactata tcaacaactg ataggagaaa caataaactc attttcaaag
     2101 tgaatttgtt agaaatggat gataaaatat tggttgactt ccggctttct aagggtgatg
     2161 gattggagtt caagagacac ttcctgaaga ttaaagggaa gctgattgat attgtgagca
     2221 gccagaagat ttggcttcct gccacatgac tcgagtctag agggcccgtt taaacccgct
     2281 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2341 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2401 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2461 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2521 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2581 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2641 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2701 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2761 accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt
     2821 cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt
     2881 ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg
     2941 agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg
     3001 taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg
     3061 ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc ctgacctatt
     3121 gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg
     3181 ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga
     3241 cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt
     3301 tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg
     3361 tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg
     3421 aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc
     3481 gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg
     3541 ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg
     3601 agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg
     3661 gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc
     3721 agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg
     3781 cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa
     3841 accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt
     3901 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3961 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4021 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4081 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4141 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4201 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4261 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4321 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4381 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4441 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4501 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4561 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4621 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4681 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4741 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4801 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4921 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4981 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5041 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5101 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5161 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5221 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5281 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5341 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5401 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5461 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5521 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5581 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5641 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5701 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5761 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5821 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5881 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5941 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6001 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6061 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6121 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6181 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6241 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6301 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6361 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6421 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6481 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgc
//
