LOCUS       sequence-221124-        7443 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 7443)
  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 7443)
  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..7443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..444
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        445..648
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     599..619
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    650..668
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    671..689
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     699..723
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      904..913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(910..912,913..915,916..1626)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(910..912,913..915,916..1626)
                     /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(955..976)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1216..1235)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1563..1584
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3610..3627)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3616..3840
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3973..3992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4124..4171
                     /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             4215..4811
                     /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     4708..4728
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4947..5067
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4983..5002)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5037..5056
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5116..5132)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5116..5132)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5129..5151)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5140..5156
                     /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(5164..5170,5171..5188,5189..5194))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5209..5230
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5347..5364)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5518..6106)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5598..5617)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6277..7068,7069..7137))
                     /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     6900..6919
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7138..7242)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7317..7336)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
       61 cgttgacatt gattattgtc tagttattaa tagtaatcaa ttacggggtc attagttcat
      121 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      181 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      241 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      301 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      361 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      421 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      481 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      541 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      601 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt
      661 gatagagatc tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag
      721 atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc
      781 agcctccgga ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca
      841 gtgtggtgga attctgcaga tatccagcac agtggcggcc gctcgagtct agcgctaccg
      901 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      961 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1021 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1081 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1141 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1201 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1261 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1321 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1381 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1441 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1501 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1561 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1621 tacaagtccg gactcagatc catgaagcgc agttcagttt ccagcggtgg tgctggccgc
     1681 ctctccatgc aggagttaag atcccaggat gtaaataaac aaggcctcta tacccctcaa
     1741 accaaagaga aaccaacctt tggaaagttg agtataaaca aaccgacatc tgaaagaaaa
     1801 gtctcgctat ttggcaaaag aactagtgga catggatcac ggaatagtca acttggtata
     1861 ttttccagtt ctgagaaaat caaggacccg agaccactta atgacaaagc attcattcag
     1921 cagtgtattc gacaactctg tgagtttctt acagaaaatg gttatgcaca taatgtgtcc
     1981 atgaaatctc tacaagctcc ctctgttaaa gacttcctga agatcttcac atttctttat
     2041 ggcttcctgt gcccctcata cgaacttcct gacacagagt ttgaagaaga ggttccagaa
     2101 atctttgaag accttgggta tccttttgca ctatccaaaa gctccatgta cacagtgggg
     2161 gctcctcata catggcctca cattgtggca gccttagttt ggctaataga ctgcatcaag
     2221 atacatactg ccatgaaaga aagctcacct ttatttgatg atgggcagcc ttggggagaa
     2281 gaaactgaag atggaattat gcataataag ttgtttttgg actacaccat aaaatgctat
     2341 gagagtttta tgagtggtgc cgacagcttt gatgagatga atgcagagct gcagtcaaaa
     2401 ctgaaggatt tatttaatgt ggatgctttt aagctggaat cattagaagc aaaaaacaga
     2461 gcattgaatg aacagattgc aagattggaa caagaaagag aaaaagaacc gaatcgtcta
     2521 gagtcgttga gaaaactgaa ggcttcctta caaggagatg ttcaaaagta tcaggcatac
     2581 atgagcaatt tggagtctca ttcagccatt cttgaccaga aattaaatgg tctcaatgag
     2641 gaaattgcta gagtagaact agaatgtgaa acaataaaac aggagaacac tcgactacag
     2701 aatatcattg acaaccagaa gtactcagtt gcagacattg agcgaataaa tcatgaaaga
     2761 aatgaattgc agcagactat taataaatta accaaggacc tggaagctga acaacagaag
     2821 ttgtggaatg aggagttaaa atatgccaga ggcaaagaag cgattgaaac acaattagca
     2881 gagtatcaca aattggctag aaaattaaaa cttattccta aaggtgctga gaattccaaa
     2941 ggttatgact ttgaaattaa gtttaatccc gaggctggtg ccaactgcct tgtcaaatac
     3001 agggctcaag tttatgtacc tcttaaggaa ctcctgaatg aaactgaaga agaaattaat
     3061 aaagccctaa ataaaaaaat gggtttggag gatactttag aacaattgaa tgcaatgata
     3121 acagaaagca agagaagtgt gagaactctg aaagaagaag ttcaaaagct ggatgatctt
     3181 taccaacaaa aaattaagga agcagaggaa gaggatgaaa aatgtgccag tgagcttgag
     3241 tccttggaga aacacaagca cctgctagaa agtactgtta accaggggct cagtgaagct
     3301 atgaatgaat tagatgctgt tcagcgggaa taccaactag ttgtgcaaac cacgactgaa
     3361 gaaagacgaa aagtgggaaa taacttgcaa cgtctgttag agatggttgc tacacatgtt
     3421 gggtctgtag agaaacatct tgaggagcag attgctaaag ttgatagaga atatgaagaa
     3481 tgcatgtcag aagatctctc ggaaaatatt aaagagatta gagataagta tgagaagaaa
     3541 gctactctaa ttaagtcttc tgaagaatga ctcgacggta ccgcgggccc gtttaaaccc
     3601 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3661 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3721 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3781 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3841 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     3901 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3961 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4021 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4081 tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa
     4141 gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga
     4201 cgtgctggcg cgccaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     4261 cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     4321 tcgacccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     4381 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga
     4441 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     4501 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     4561 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca
     4621 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     4681 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     4741 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     4801 ccggtgcctg acctaggcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4861 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4921 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttaaatg gttacaaata
     4981 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5041 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5101 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5161 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5221 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5281 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5341 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5401 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5461 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5521 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5581 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5641 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5701 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5761 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5821 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5881 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5941 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     6001 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6061 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6121 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6181 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6241 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6301 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6361 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6421 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6481 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6541 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6601 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6661 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6721 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6781 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6841 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6901 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6961 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     7021 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     7081 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     7141 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7201 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7261 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     7321 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     7381 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
     7441 atg
//
