LOCUS       sequence-221039-        7828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of eGFP-tagged HEC1-9A 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-HEC1-9A
FEATURES             Location/Qualifiers
     source          1..7828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        152..531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        532..735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     686..706
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    737..755
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    758..776
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     786..810
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      991..1000
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(997..999,1000..1002,1003..1713)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(997..999,1000..1002,1003..1713)
                     /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(1042..1063)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1303..1322)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1650..1671
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3697..3714)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3703..3927
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4060..4079)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4211..4258
                     /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             4266..5288
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4266..5288
                     /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    5418..5539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5455..5474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5509..5528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5588..5604)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5588..5604)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5601..5623)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5612..5628
                     /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(5636..5642,5643..5660,5661..5666))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5681..5702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5819..5836)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5990..6578)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6070..6089)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6749..7540,7541..7609))
                     /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     7372..7391
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7610..7714)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7789..7808)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
       61 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      121 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      541 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      661 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      721 tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt
      781 cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac
      841 ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa acttaagctt
      901 ggtaccgagc tcggatccac tagtccagtg tggtggaatt ctgcagatat ccagcacagt
      961 ggcggccgct cgagtctagc gctaccggtc gccaccatgg tgagcaaggg cgaggagctg
     1021 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1081 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1141 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1201 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1261 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1321 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1381 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1441 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     1501 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1561 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1621 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1681 gggatcactc tcggcatgga cgagctgtac aagtccggac tcagatccat gaagcgcgct
     1741 gcagtttccg ccggtggtgc tggccgcctc gccatgcagg agttaagatc ccaggatgta
     1801 aataaacaag gcctctatac ccctcaaacc aaagagaaac caacctttgg aaagttggct
     1861 ataaacaaac cggcatctga aagaaaagtc gcgctatttg gcaaaagaac tgctggacat
     1921 ggatcccgga atgctcaact tggtatattt tccagttctg agaaaatcaa ggacccgaga
     1981 ccacttaacg acaaagcatt cattcagcag tgtattcgac aactctgtga gtttcttaca
     2041 gaaaatggtt atgcacataa tgtgtccatg aaatctctac aagctccctc tgttaaagac
     2101 ttcctgaaga tcttcacatt tctttatggc ttcctgtgcc cctcatacga acttcctgac
     2161 acaaagtttg aagaagaggt tccaagaatc tttaaagacc ttgggtatcc ttttgcacta
     2221 tccaaaagct ccatgtacac agtgggggct cctcatacat ggcctcacat tgtggcagcc
     2281 ttagtttggc taatagactg catcaagata catactgcca tgaaagaaag ctcaccttta
     2341 tttgatgatg ggcagccttg gggagaagaa actgaagatg gaattatgca taataagttg
     2401 tttttggact acaccataaa atgctatgag agttttatga gtggtgccga cagctttgat
     2461 gagatgaatg cagagctgca gtcaaaactg aaggatttat ttaatgtgga tgcttttaag
     2521 ctggaatcat tagaagcaaa aaacagagca ttgaatgaac agattgcaag attggaacaa
     2581 gaaagagaaa aagaaccgaa tcgtctagag tcgttgagaa aactgaaggc ttccttacaa
     2641 ggagatgttc aaaagtatca ggcatacatg agcaatttgg agtctcattc agccattctt
     2701 gaccagaaat taaatggtct caatgaggaa attgctagag tagaactaga atgtgaaaca
     2761 ataaaacagg agaacactcg actacagaat atcattgaca accagaagta ctcagttgca
     2821 gacattgagc gaataaatca tgaaagaaat gaattgcagc agactattaa taaattaacc
     2881 aaggacctgg aagctgaaca acagaagttg tggaatgagg agttaaaata tgccagaggc
     2941 aaagaagcga ttgaaacaca attagcagag tatcacaaat tggctagaaa attaaaactt
     3001 attcctaaag gtgctgagaa ttccaaaggt tatgactttg aaattaagtt taatcccgag
     3061 gctggtgcca actgccttgt caaatacagg gctcaagttt atgtacctct taaggaactc
     3121 ctgaatgaaa ctgaagaaga aattaataaa gccctaaata aaaaaatggg tttggaggat
     3181 actttagaac aattgaatgc aatgataaca gaaagcaaga gaagtgtgag aactctgaaa
     3241 gaagaagttc aaaaattgga tgatctttac caacaaaaaa ttaaggaagc agaggaagag
     3301 gatgaaaaat gtgccagtga gcttgagtcc ttggagaaac acaagcacct gctagaaagt
     3361 actgttaacc aggggctcag tgaagctatg aatgaattag atgctgttca gcgggaatac
     3421 caactagttg tgcaaaccac gactgaagaa agacgaaaag tgggaaataa cttgcaacgt
     3481 ctgttagaga tggttgctac acatgttggg tctgtagaga aacatcttga ggagcagatt
     3541 gctaaagttg atagagaata tgaagaatgc atgtcagaag atctctcgga aaatattaaa
     3601 gagattagag ataagtatga gaagaaagct actctaatta agtcttctga agaatgactc
     3661 gacggtaccg cgggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3721 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3781 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3841 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3901 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3961 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4021 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4081 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4141 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4201 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     4261 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     4321 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     4381 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     4441 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     4501 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     4561 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     4621 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     4681 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     4741 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     4801 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     4861 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     4921 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     4981 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     5041 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     5101 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     5161 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     5221 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     5281 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5341 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5401 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5461 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5521 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5581 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5641 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5701 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5761 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5821 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5881 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5941 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6001 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6061 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6121 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6181 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6241 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6301 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6361 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6421 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6481 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6541 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6601 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6661 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6721 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6781 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6841 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6901 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6961 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7021 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7081 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7141 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7201 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7261 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7321 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7381 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7441 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7501 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7561 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7621 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7681 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     7741 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
     7801 tgatgccgca tagttaagcc agtatctg
//
