LOCUS       sequence-221043-        7828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of eGFP-tagged HEC1-9D 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-HEC1-9D
FEATURES             Location/Qualifiers
     source          1..7828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        443..646
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     597..617
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    648..666
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    669..687
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     697..721
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      902..911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(908..910,911..913,914..1624)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(908..910,911..913,914..1624)
                     /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(953..974)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1214..1233)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1561..1582
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3608..3625)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3614..3838
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3971..3990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4122..4169
                     /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             4177..5199
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4177..5199
                     /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    5329..5450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5366..5385)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5420..5439
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5499..5515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5499..5515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5512..5534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5523..5539
                     /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(5547..5553,5554..5571,5572..5577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5592..5613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5730..5747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5901..6489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5981..6000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6660..7451,7452..7520))
                     /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     7283..7302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7521..7625)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7700..7719)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
       61 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      121 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      181 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      241 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      301 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      361 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      421 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      481 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      541 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      601 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
      661 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
      721 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      781 cctccggact ctagcgttta aacttaagct tggtaccgag ctcggatcca ctagtccagt
      841 gtggtggaat tctgcagata tccagcacag tggcggccgc tcgagtctag cgctaccggt
      901 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
      961 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1021 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1081 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     1141 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1201 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1261 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1321 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     1381 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     1441 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     1501 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     1561 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1621 caagtccgga ctcagatcca tgaagcgcga tgatgtttcc gacggtggtg ctggccgcct
     1681 cgacatgcag gagttaagat cccaggatgt aaataaacaa ggcctctata cccctcaaac
     1741 caaagagaaa ccaacctttg gaaagttgga tataaacaaa ccggactctg aaagaaaagt
     1801 cgacctattt ggcaaaagaa ctgatggaca tggatcccgg aatgatcaac ttggtatatt
     1861 ttccagttct gagaaaatca aggacccgag accacttaac gacaaagcat tcattcagca
     1921 gtgtattcga caactctgtg agtttcttac agaaaatggt tatgcacata atgtgtccat
     1981 gaaatctcta caagctccct ctgttaaaga cttcctgaag atcttcacat ttctttatgg
     2041 cttcctgtgc ccctcatacg aacttcctga cacaaagttt gaagaagagg ttccaagaat
     2101 ctttaaagac cttgggtatc cttttgcact atccaaaagc tccatgtaca cagtgggggc
     2161 tcctcataca tggcctcaca ttgtggcagc cttagtttgg ctaatagact gcatcaagat
     2221 acatactgcc atgaaagaaa gctcaccttt atttgatgat gggcagcctt ggggagaaga
     2281 aactgaagat ggaattatgc ataataagtt gtttttggac tacaccataa aatgctatga
     2341 gagttttatg agtggtgccg acagctttga tgagatgaat gcagagctgc agtcaaaact
     2401 gaaggattta tttaatgtgg atgcttttaa gctggaatca ttagaagcaa aaaacagagc
     2461 attgaatgaa cagattgcaa gattggaaca agaaagagaa aaagaaccga atcgtctaga
     2521 gtcgttgaga aaactgaagg cttccttaca aggagatgtt caaaagtatc aggcatacat
     2581 gagcaatttg gagtctcatt cagccattct tgaccagaaa ttaaatggtc tcaatgagga
     2641 aattgctaga gtagaactag aatgtgaaac aataaaacag gagaacactc gactacagaa
     2701 tatcattgac aaccagaagt actcagttgc agacattgag cgaataaatc atgaaagaaa
     2761 tgaattgcag cagactatta ataaattaac caaggacctg gaagctgaac aacagaagtt
     2821 gtggaatgag gagttaaaat atgccagagg caaagaagcg attgaaacac aattagcaga
     2881 gtatcacaaa ttggctagaa aattaaaact tattcctaaa ggtgctgaga attccaaagg
     2941 ttatgacttt gaaattaagt ttaatcccga ggctggtgcc aactgccttg tcaaatacag
     3001 ggctcaagtt tatgtacctc ttaaggaact cctgaatgaa actgaagaag aaattaataa
     3061 agccctaaat aaaaaaatgg gtttggagga tactttagaa caattgaatg caatgataac
     3121 agaaagcaag agaagtgtga gaactctgaa agaagaagtt caaaaattgg atgatcttta
     3181 ccaacaaaaa attaaggaag cagaggaaga ggatgaaaaa tgtgccagtg agcttgagtc
     3241 cttggagaaa cacaagcacc tgctagaaag tactgttaac caggggctca gtgaagctat
     3301 gaatgaatta gatgctgttc agcgggaata ccaactagtt gtgcaaacca cgactgaaga
     3361 aagacgaaaa gtgggaaata acttgcaacg tctgttagag atggttgcta cacatgttgg
     3421 gtctgtagag aaacatcttg aggagcagat tgctaaagtt gatagagaat atgaagaatg
     3481 catgtcagaa gatctctcgg aaaatattaa agagattaga gataagtatg agaagaaagc
     3541 tactctaatt aagtcttctg aagaatgact cgacggtacc gcgggcccgt ttaaacccgc
     3601 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3661 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3721 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3781 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3841 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3901 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3961 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4021 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4081 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     4141 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     4201 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     4261 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     4321 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     4381 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     4441 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     4501 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     4561 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     4621 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     4681 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     4741 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     4801 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     4861 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     4921 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     4981 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     5041 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     5101 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     5161 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     5221 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5281 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     5341 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5401 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     5461 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5521 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5581 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5641 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5701 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5761 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5821 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5881 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5941 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6001 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6061 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6121 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6181 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6241 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6301 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6361 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6421 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6481 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6541 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6601 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6661 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6721 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6781 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6841 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6901 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6961 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7021 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7081 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7141 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7201 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7261 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7321 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7381 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7441 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7501 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7561 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7621 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     7681 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     7741 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     7801 ggcaaggctt gaccgacaat tgcatgaa
//
