LOCUS       sequence-221035-        7828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of eGFP-tagged HEC1 in FLPin Trex cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7828)
  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 7828)
  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-hygro-eGFP-HEC1
FEATURES             Location/Qualifiers
     source          1..7828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    675..693
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    696..714
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     724..748
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      929..938
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(935..937,938..940,941..1651)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(935..937,938..940,941..1651)
                     /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(980..1001)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1241..1260)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1588..1609
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3635..3652)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3641..3865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3998..4017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4149..4196
                     /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             4204..5226
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4204..5226
                     /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    5356..5477
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5393..5412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5447..5466
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5526..5542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5526..5542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5539..5561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5550..5566
                     /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(5574..5580,5581..5598,5599..5604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5619..5640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5757..5774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5928..6516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6008..6027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6687..7478,7479..7547))
                     /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     7310..7329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7548..7652)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7727..7746)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
       61 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctctcccta tcagtgatag agatctccct atcagtgata gagatcgtcg
      721 acgagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      781 ccatagaaga caccgggacc gatccagcct ccggactcta gcgtttaaac ttaagcttgg
      841 taccgagctc ggatccacta gtccagtgtg gtggaattct gcagatatcc agcacagtgg
      901 cggccgctcg agtctagcgc taccggtcgc caccatggtg agcaagggcg aggagctgtt
      961 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1021 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1081 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1141 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1201 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1261 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1321 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     1381 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1441 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1501 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1561 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1621 gatcactctc ggcatggacg agctgtacaa gtccggactc agatccatga agcgcagttc
     1681 agtttccagc ggtggtgctg gccgcctctc catgcaggag ttaagatccc aggatgtaaa
     1741 taaacaaggc ctctataccc ctcaaaccaa agagaaacca acctttggaa agttgagtat
     1801 aaacaaaccg acatctgaaa gaaaagtctc gctatttggc aaaagaacta gtggacatgg
     1861 atcacggaat agtcaacttg gtatattttc cagttctgag aaaatcaagg acccgagacc
     1921 acttaacgac aaagcattca ttcagcagtg tattcgacaa ctctgtgagt ttcttacaga
     1981 aaatggttat gcacataatg tgtccatgaa atctctacaa gctccctctg ttaaagactt
     2041 cctgaagatc ttcacatttc tttatggctt cctgtgcccc tcatacgaac ttcctgacac
     2101 aaagtttgaa gaagaggttc caagaatctt taaagacctt gggtatcctt ttgcactatc
     2161 caaaagctcc atgtacacag tgggggctcc tcatacatgg cctcacattg tggcagcctt
     2221 agtttggcta atagactgca tcaagataca tactgccatg aaagaaagct cacctttatt
     2281 tgatgatggg cagccttggg gagaagaaac tgaagatgga attatgcata ataagttgtt
     2341 tttggactac accataaaat gctatgagag ttttatgagt ggtgccgaca gctttgatga
     2401 gatgaatgca gagctgcagt caaaactgaa ggatttattt aatgtggatg cttttaagct
     2461 ggaatcatta gaagcaaaaa acagagcatt gaatgaacag attgcaagat tggaacaaga
     2521 aagagaaaaa gaaccgaatc gtctagagtc gttgagaaaa ctgaaggctt ccttacaagg
     2581 agatgttcaa aagtatcagg catacatgag caatttggag tctcattcag ccattcttga
     2641 ccagaaatta aatggtctca atgaggaaat tgctagagta gaactagaat gtgaaacaat
     2701 aaaacaggag aacactcgac tacagaatat cattgacaac cagaagtact cagttgcaga
     2761 cattgagcga ataaatcatg aaagaaatga attgcagcag actattaata aattaaccaa
     2821 ggacctggaa gctgaacaac agaagttgtg gaatgaggag ttaaaatatg ccagaggcaa
     2881 agaagcgatt gaaacacaat tagcagagta tcacaaattg gctagaaaat taaaacttat
     2941 tcctaaaggt gctgagaatt ccaaaggtta tgactttgaa attaagttta atcccgaggc
     3001 tggtgccaac tgccttgtca aatacagggc tcaagtttat gtacctctta aggaactcct
     3061 gaatgaaact gaagaagaaa ttaataaagc cctaaataaa aaaatgggtt tggaggatac
     3121 tttagaacaa ttgaatgcaa tgataacaga aagcaagaga agtgtgagaa ctctgaaaga
     3181 agaagttcaa aagctggatg atctttacca acaaaaaatt aaggaagcag aggaagagga
     3241 tgaaaaatgt gccagtgagc ttgagtcctt ggagaaacac aagcacctgc tagaaagtac
     3301 tgttaaccag gggctcagtg aagctatgaa tgaattagat gctgttcagc gggaatacca
     3361 actagttgtg caaaccacga ctgaagaaag acgaaaagtg ggaaataact tgcaacgtct
     3421 gttagagatg gttgctacac atgttgggtc tgtagagaaa catcttgagg agcagattgc
     3481 taaagttgat agagaatatg aagaatgcat gtcagaagat ctctcggaaa atattaaaga
     3541 gattagagat aagtatgaga agaaagctac tctaattaag tcttctgaag aatgactcga
     3601 cggtaccgcg ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     3661 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3721 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3781 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3841 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3901 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3961 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4021 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4081 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4141 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     4201 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     4261 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     4321 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     4381 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     4441 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     4501 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     4561 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     4621 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     4681 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     4741 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     4801 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     4861 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     4921 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     4981 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     5041 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     5101 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     5161 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     5221 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5281 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5341 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5401 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5461 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     5521 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5581 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5641 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5701 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5761 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5821 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5881 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5941 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     6001 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     6061 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6121 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6181 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6241 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6301 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6361 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6421 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6481 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6541 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6601 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6661 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6721 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6781 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6841 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6901 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6961 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     7021 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     7081 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     7141 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     7201 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     7261 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7321 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7381 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7441 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7501 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7561 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7621 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7681 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     7741 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     7801 gcgcgagcaa aatttaagct acaacaag
//
