LOCUS       sequence-221121-        7443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HEC1 M2 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-M2
FEATURES             Location/Qualifiers
     source          1..7443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        69..448
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        449..652
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     603..623
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    654..672
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    675..693
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     703..727
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      908..917
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(914..916,917..919,920..1630)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(914..916,917..919,920..1630)
                     /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(959..980)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1220..1239)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1567..1588
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3614..3631)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3620..3844
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3977..3996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4128..4175
                     /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             4219..4815
                     /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     4712..4732
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4951..5071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4987..5006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5041..5060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5120..5136)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5120..5136)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5133..5155)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5144..5160
                     /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(5168..5174,5175..5192,5193..5198))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5213..5234
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5351..5368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5522..6110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5602..5621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6281..7072,7073..7141))
                     /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     6904..6923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7142..7246)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7321..7340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
       61 tacgcgttga cattgattat tgtctagtta ttaatagtaa tcaattacgg ggtcattagt
      121 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      181 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      241 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      301 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      361 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      421 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      481 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      541 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      601 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctccctat
      661 cagtgataga gatctcccta tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg
      721 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
      781 atccagcctc cggactctag cgtttaaact taagcttggt accgagctcg gatccactag
      841 tccagtgtgg tggaattctg cagatatcca gcacagtggc ggccgctcga gtctagcgct
      901 accggtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
      961 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     1021 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1081 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     1141 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1201 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     1261 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1321 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     1381 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     1441 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1501 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     1561 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     1621 gctgtacaag tccggactca gatccatgaa gcgcagttca gtttccagcg gtggtgctgg
     1681 ccgcctctcc atgcaggagt taagatccca ggatgtaaat aaacaaggcc tctatacccc
     1741 tcaaaccaaa gagaaaccaa cctttggaaa gttgagtata aacaaaccga catctgaaag
     1801 aaaagtctcg ctatttggca aaagaactag tggacatgga tcacggaata gtcaacttgg
     1861 tatattttcc agttctgaga aaatcgagga cccgagacca cttgaagacg aagcattcat
     1921 tcagcagtgt attcgacaac tctgtgagtt tcttacagaa aatggttatg cacataatgt
     1981 gtccatgaaa tctctacaag ctccctctgt taaagacttc ctgaagatct tcacatttct
     2041 ttatggcttc ctgtgcccct catacgaact tcctgacaca aagtttgaag aagaggttcc
     2101 aagaatcttt aaagaccttg ggtatccttt tgcactatcc aaaagctcca tgtacacagt
     2161 gggggctcct catacatggc ctcacattgt ggcagcctta gtttggctaa tagactgcat
     2221 caagatacat actgccatga aagaaagctc acctttattt gatgatgggc agccttgggg
     2281 agaagaaact gaagatggaa ttatgcataa taagttgttt ttggactaca ccataaaatg
     2341 ctatgagagt tttatgagtg gtgccgacag ctttgatgag atgaatgcag agctgcagtc
     2401 aaaactgaag gatttattta atgtggatgc ttttaagctg gaatcattag aagcaaaaaa
     2461 cagagcattg aatgaacaga ttgcaagatt ggaacaagaa agagaaaaag aaccgaatcg
     2521 tctagagtcg ttgagaaaac tgaaggcttc cttacaagga gatgttcaaa agtatcaggc
     2581 atacatgagc aatttggagt ctcattcagc cattcttgac cagaaattaa atggtctcaa
     2641 tgaggaaatt gctagagtag aactagaatg tgaaacaata aaacaggaga acactcgact
     2701 acagaatatc attgacaacc agaagtactc agttgcagac attgagcgaa taaatcatga
     2761 aagaaatgaa ttgcagcaga ctattaataa attaaccaag gacctggaag ctgaacaaca
     2821 gaagttgtgg aatgaggagt taaaatatgc cagaggcaaa gaagcgattg aaacacaatt
     2881 agcagagtat cacaaattgg ctagaaaatt aaaacttatt cctaaaggtg ctgagaattc
     2941 caaaggttat gactttgaaa ttaagtttaa tcccgaggct ggtgccaact gccttgtcaa
     3001 atacagggct caagtttatg tacctcttaa ggaactcctg aatgaaactg aagaagaaat
     3061 taataaagcc ctaaataaaa aaatgggttt ggaggatact ttagaacaat tgaatgcaat
     3121 gataacagaa agcaagagaa gtgtgagaac tctgaaagaa gaagttcaaa agctggatga
     3181 tctttaccaa caaaaaatta aggaagcaga ggaagaggat gaaaaatgtg ccagtgagct
     3241 tgagtccttg gagaaacaca agcacctgct agaaagtact gttaaccagg ggctcagtga
     3301 agctatgaat gaattagatg ctgttcagcg ggaataccaa ctagttgtgc aaaccacgac
     3361 tgaagaaaga cgaaaagtgg gaaataactt gcaacgtctg ttagagatgg ttgctacaca
     3421 tgttgggtct gtagagaaac atcttgagga gcagattgct aaagttgata gagaatatga
     3481 agaatgcatg tcagaagatc tctcggaaaa tattaaagag attagagata agtatgagaa
     3541 gaaagctact ctaattaagt cttctgaaga atgactcgac ggtaccgcgg gcccgtttaa
     3601 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3661 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3721 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3781 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3841 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3901 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3961 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4021 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4081 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     4141 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     4201 gcgacgtgct ggcgcgccac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4261 gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4321 accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4381 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4441 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4501 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     4561 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     4621 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     4681 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     4741 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     4801 aagcccggtg cctgacctag gcacgtacta cgagatttcg attccaccgc cgccttctat
     4861 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4921 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta aatggttaca
     4981 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5041 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5101 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5161 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5221 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5281 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5341 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5401 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5461 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5521 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5581 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5641 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5701 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5761 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5821 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5881 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5941 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6001 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6061 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6121 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6181 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6241 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6301 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6361 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6421 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6481 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6541 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6601 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6661 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6721 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6781 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6841 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6901 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6961 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7021 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7081 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7141 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7201 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7261 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     7321 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     7381 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     7441 ttg
//
