LOCUS       sequence-221024-        7203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of eGFP-tagged HEC1-d80 in FLPin Trex cell 
            lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7203)
  AUTHORS   Etemad B, Kuijt TE, Kops GJ
  TITLE     Kinetochore-microtubule attachment is sufficient to satisfy the 
            human spindle assembly checkpoint.
  JOURNAL   Nat Commun. 2015 Dec 1;6:8987. doi: 10.1038/ncomms9987.
  PUBMED    26621779
REFERENCE   2  (bases 1 to 7203)
  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: "Nat 
            Commun."; date: "2015-12-1"; volume: "6:8987. doi"; pages: " 
            10.1038/ncomms9987"
COMMENT     Sequence Label: pcDNA5-puro-eGFP-HEC1-d80
FEATURES             Location/Qualifiers
     source          1..7203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        131..510
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..714
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     665..685
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    716..734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    737..755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     765..789
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      970..979
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(976..978,979..981,982..1692)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(976..978,979..981,982..1692)
                     /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(1021..1042)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1282..1301)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1629..1650
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3436..3453)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3442..3666
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3799..3818)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3950..3997
                     /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             4041..4637
                     /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     4534..4554
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4773..4893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4809..4828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4863..4882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4942..4958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4942..4958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4955..4977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4966..4982
                     /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(4990..4996,4997..5014,5015..5020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5035..5056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5173..5190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5344..5932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5424..5443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6103..6894,6895..6963))
                     /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     6726..6745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6964..7068)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7143..7162)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
       61 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      121 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      181 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      241 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      301 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      361 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      421 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      481 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      541 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      601 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      661 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct
      721 atcagtgata gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac
      781 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
      841 cgatccagcc tccggactct agcgtttaaa cttaagcttg gtaccgagct cggatccact
      901 agtccagtgt ggtggaattc tgcagatatc cagcacagtg gcggccgctc gagtctagcg
      961 ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1021 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1081 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1141 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1201 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1261 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1321 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1381 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1441 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1501 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1561 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1621 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1681 gagctgtaca agtccggact cagatccaag gacccgagac cacttaacga caaagcattc
     1741 attcagcagt gtattcgaca actctgtgag tttcttacag aaaatggtta tgcacataat
     1801 gtgtccatga aatctctaca agctccctct gttaaagact tcctgaagat cttcacattt
     1861 ctttatggct tcctgtgccc ctcatacgaa cttcctgaca caaagtttga agaagaggtt
     1921 ccaagaatct ttaaagacct tgggtatcct tttgcactat ccaaaagctc catgtacaca
     1981 gtgggggctc ctcatacatg gcctcacatt gtggcagcct tagtttggct aatagactgc
     2041 atcaagatac atactgccat gaaagaaagc tcacctttat ttgatgatgg gcagccttgg
     2101 ggagaagaaa ctgaagatgg aattatgcat aataagttgt ttttggacta caccataaaa
     2161 tgctatgaga gttttatgag tggtgccgac agctttgatg agatgaatgc agagctgcag
     2221 tcaaaactga aggatttatt taatgtggat gcttttaagc tggaatcatt agaagcaaaa
     2281 aacagagcat tgaatgaaca gattgcaaga ttggaacaag aaagagaaaa agaaccgaat
     2341 cgtctagagt cgttgagaaa actgaaggct tccttacaag gagatgttca aaagtatcag
     2401 gcatacatga gcaatttgga gtctcattca gccattcttg accagaaatt aaatggtctc
     2461 aatgaggaaa ttgctagagt agaactagaa tgtgaaacaa taaaacagga gaacactcga
     2521 ctacagaata tcattgacaa ccagaagtac tcagttgcag acattgagcg aataaatcat
     2581 gaaagaaatg aattgcagca gactattaat aaattaacca aggacctgga agctgaacaa
     2641 cagaagttgt ggaatgagga gttaaaatat gccagaggca aagaagcgat tgaaacacaa
     2701 ttagcagagt atcacaaatt ggctagaaaa ttaaaactta ttcctaaagg tgctgagaat
     2761 tccaaaggtt atgactttga aattaagttt aatcccgagg ctggtgccaa ctgccttgtc
     2821 aaatacaggg ctcaagttta tgtacctctt aaggaactcc tgaatgaaac tgaagaagaa
     2881 attaataaag ccctaaataa aaaaatgggt ttggaggata ctttagaaca attgaatgca
     2941 atgataacag aaagcaagag aagtgtgaga actctgaaag aagaagttca aaagctggat
     3001 gatctttacc aacaaaaaat taaggaagca gaggaagagg atgaaaaatg tgccagtgag
     3061 cttgagtcct tggagaaaca caagcacctg ctagaaagta ctgttaacca ggggctcagt
     3121 gaagctatga atgaattaga tgctgttcag cgggaatacc aactagttgt gcaaaccacg
     3181 actgaagaaa gacgaaaagt gggaaataac ttgcaacgtc tgttagagat ggttgctaca
     3241 catgttgggt ctgtagagaa acatcttgag gagcagattg ctaaagttga tagagaatat
     3301 gaagaatgca tgtcagaaga tctctcggaa aatattaaag agattagaga taagtatgag
     3361 aagaaagcta ctctaattaa gtcttctgaa gaatgactcg acggtaccgc gggcccgttt
     3421 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     3481 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     3541 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     3601 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     3661 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     3721 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3781 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3841 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3901 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat
     3961 tccgaagttc ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca
     4021 ccgcgacgtg ctggcgcgcc accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     4081 acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     4141 acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     4201 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     4261 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     4321 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     4381 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     4441 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     4501 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     4561 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4621 gcaagcccgg tgcctgacct aggcacgtac tacgagattt cgattccacc gccgccttct
     4681 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4741 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct taaatggtta
     4801 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4861 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4921 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4981 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5041 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5101 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5161 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5221 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5761 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6061 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6241 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7081 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc
     7141 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg
     7201 gag
//
