LOCUS       sequence-215378-        7405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HEC1 M4 mutant tagged with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7405)
  AUTHORS   Hiruma Y, Sacristan C, Pachis ST, Adamopoulos A, Kuijt T, Ubbink M, 
            von Castelmur E, Perrakis A, Kops GJ
  TITLE     CELL DIVISION CYCLE. Competition between MPS1 and microtubules at 
            kinetochores regulates spindle checkpoint signaling.
  JOURNAL   Science. 2015 Jun 12;348(6240):1264-7. doi: 10.1126/science.aaa4055.
            Epub 2015 Jun 11.
  PUBMED    26068855
REFERENCE   2  (bases 1 to 7405)
  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: 
            "10.1126/science.aaa4055"; journalName: "Science"; date: 
            "2015-06-12- 12"; volume: "348"; issue: "6240"; pages: "1264-7"
COMMENT     Sequence Label: pcDNA5-puro-eGFP-HEC1-M4
FEATURES             Location/Qualifiers
     source          1..7405
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1074..1083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1080..1082,1083..1085,1086..1796)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1080..1082,1083..1085,1086..1796)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1125..1146)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1386..1405)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1733..1754
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3783..3800)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3789..4013
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4146..4165)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4297..4344
                     /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             4352..4948
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4845..4865
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5078..5199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5115..5134)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5169..5188
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5248..5264)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5248..5264)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5261..5283)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5272..5288
                     /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(5296..5302,5303..5320,5321..5326))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5341..5362
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5479..5496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5650..6238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5730..5749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6409..7200,7201..7269))
                     /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     7032..7051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7270..7374)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcac agtggcggcc gctcgagtct agcgctaccg gtcgccacca
     1081 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1141 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1201 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1261 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1321 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1381 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1441 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1501 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1561 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1621 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1681 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1741 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg
     1801 gactcagatc catgaagcgc agttcagttt ccagcggtgg tgctggccgc ctctccatgc
     1861 aggagttaag atcccaggat gtaaataaac aaggcctcta tacccctcaa accaaagaga
     1921 aaccaacctt tggaaagttg agtataaaca aaccgacatc tgaaagaaaa gtctcgctat
     1981 ttggcaaaag aactagtgga catggatccc ggaatagtca acttggtata ttttccagtt
     2041 ctgagaaaat caaggacccg agaccactta acgacaaagc attcattcag cagtgtattc
     2101 gacaactctg tgagtttctt acagaaaatg gttatgcaca taatgtgtcc atgaaatctc
     2161 tacaagctcc ctctgttaaa gacttcctga agatcttcac atttctttat ggcttcctgt
     2221 gcccctcata cgaacttcct gacacagagt ttgaagaaga ggttccagaa atctttgaag
     2281 accttgggta tccttttgca ctatccaaaa gctccatgta cacagtgggg gctcctcata
     2341 catggcctca cattgtggca gccttagttt ggctaataga ctgcatcaag atacatactg
     2401 ccatgaaaga aagctcacct ttatttgatg atgggcagcc ttggggagaa gaaactgaag
     2461 atggaattat gcataataag ttgtttttgg actacaccat aaaatgctat gagagtttta
     2521 tgagtggtgc cgacagcttt gatgagatga atgcagagct gcagtcaaaa ctgaaggatt
     2581 tatttaatgt ggatgctttt aagctggaat cattagaagc aaaaaacaga gcattgaatg
     2641 aacagattgc aagattggaa caagaaagag aaaaagaacc gaatcgtcta gagtcgttga
     2701 gaaaactgaa ggcttcctta caaggagatg ttcaaaagta tcaggcatac atgagcaatt
     2761 tggagtctca ttcagccatt cttgaccaga aattaaatgg tctcaatgag gaaattgcta
     2821 gagtagaact agaatgtgaa acaataaaac aggagaacac tcgactacag aatatcattg
     2881 acaaccagaa gtactcagtt gcagacattg agcgaataaa tcatgaaaga aatgaattgc
     2941 agcagactat taataaatta accaaggacc tggaagctga acaacagaag ttgtggaatg
     3001 aggagttaaa atatgccaga ggcaaagaag cgattgaaac acaattagca gagtatcaca
     3061 aattggctag aaaattaaaa cttattccta aaggtgctga gaattccaaa ggttatgact
     3121 ttgaaattaa gtttaatccc gaggctggtg ccaactgcct tgtcaaatac agggctcaag
     3181 tttatgtacc tcttaaggaa ctcctgaatg aaactgaaga agaaattaat aaagccctaa
     3241 ataaaaaaat gggtttggag gatactttag aacaattgaa tgcaatgata acagaaagca
     3301 agagaagtgt gagaactctg aaagaagaag ttcaaaaatt ggatgatctt taccaacaaa
     3361 aaattaagga agcagaggaa gaggatgaaa aatgtgccag tgagcttgag tccttggaga
     3421 aacacaagca cctgctagaa agtactgtta accaggggct cagtgaagct atgaatgaat
     3481 tagatgctgt tcagcgggaa taccaactag ttgtgcaaac cacgactgaa gaaagacgaa
     3541 aagtgggaaa taacttgcaa cgtctgttag agatggttgc tacacatgtt gggtctgtag
     3601 agaaacatct tgaggagcag attgctaaag ttgatagaga atatgaagaa tgcatgtcag
     3661 aagatctctc ggaaaatatt aaagagatta gagataagta tgagaagaaa gctactctaa
     3721 ttaagtcttc tgaagaatga ctcgaggacg gtaccgcggg cccgtttaaa cccgctgatc
     3781 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3841 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3901 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3961 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4021 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     4081 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4141 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4201 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4261 caaaaaactt gattagggtg atggttcacg tacctagaag ttcctattcc gaagttccta
     4321 ttctctagaa agtataggaa cttccttggc caccgagtac aagcccacgg tgcgcctcgc
     4381 cacccgcgac gacgtccccc gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     4441 cgccacgcgc cacaccgtcg acccggaccg ccacatcgag cgggtcaccg agctgcaaga
     4501 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     4561 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     4621 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     4681 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     4741 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     4801 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     4861 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     4921 gtgcatgacc cgcaagcccg gtgcctgaca cgtactacga gatttcgatt ccaccgccgc
     4981 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     5041 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     5101 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     5161 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     5221 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5281 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     5341 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5401 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5461 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5521 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5581 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5641 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5701 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5761 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     5821 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     5881 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5941 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6001 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6061 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6121 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6181 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6241 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6301 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6361 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6421 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6481 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6541 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6601 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6661 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6721 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6781 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     6841 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6901 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6961 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7021 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7081 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7141 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7201 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7261 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7321 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7381 tccccgaaaa gtgccacctg acgtc
//
