LOCUS       sequence-221015-        7443 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 7443)
  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 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"; journalName: "Nat 
            Commun."; date: "2015-12-1"; volume: "6:8987. doi"; pages: " 
            10.1038/ncomms9987"
COMMENT     Sequence Label: pcDNA5-puro-eGFP-HEC1-9A
FEATURES             Location/Qualifiers
     source          1..7443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        41..420
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        421..624
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     575..595
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    626..644
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    647..665
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     675..699
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      880..889
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(886..888,889..891,892..1602)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(886..888,889..891,892..1602)
                     /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(931..952)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1192..1211)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1539..1560
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3586..3603)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3592..3816
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3949..3968)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4100..4147
                     /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             4191..4787
                     /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     4684..4704
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4923..5043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4959..4978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5013..5032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5092..5108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5092..5108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5105..5127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5116..5132
                     /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(5140..5146,5147..5164,5165..5170))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5185..5206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5323..5340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5494..6082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5574..5593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6253..7044,7045..7113))
                     /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     6876..6895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7114..7218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7293..7312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
       61 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      121 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      181 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      241 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      301 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      361 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      421 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      481 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      541 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      601 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      661 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      721 tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa
      781 cttaagcttg gtaccgagct cggatccact agtccagtgt ggtggaattc tgcagatatc
      841 cagcacagtg gcggccgctc gagtctagcg ctaccggtcg ccaccatggt gagcaagggc
      901 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      961 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1021 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1081 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1141 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1201 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1261 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1321 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1381 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1441 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1501 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1561 accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact cagatccatg
     1621 aagcgcgctg cagtttccgc cggtggtgct ggccgcctcg ccatgcagga gttaagatcc
     1681 caggatgtaa ataaacaagg cctctatacc cctcaaacca aagagaaacc aacctttgga
     1741 aagttggcta taaacaaacc ggcatctgaa agaaaagtcg cgctatttgg caaaagaact
     1801 gctggacatg gatcccggaa tgctcaactt ggtatatttt ccagttctga gaaaatcaag
     1861 gacccgagac cacttaacga caaagcattc attcagcagt gtattcgaca actctgtgag
     1921 tttcttacag aaaatggtta tgcacataat gtgtccatga aatctctaca agctccctct
     1981 gttaaagact tcctgaagat cttcacattt ctttatggct tcctgtgccc ctcatacgaa
     2041 cttcctgaca caaagtttga agaagaggtt ccaagaatct ttaaagacct tgggtatcct
     2101 tttgcactat ccaaaagctc catgtacaca gtgggggctc ctcatacatg gcctcacatt
     2161 gtggcagcct tagtttggct aatagactgc atcaagatac atactgccat gaaagaaagc
     2221 tcacctttat ttgatgatgg gcagccttgg ggagaagaaa ctgaagatgg aattatgcat
     2281 aataagttgt ttttggacta caccataaaa tgctatgaga gttttatgag tggtgccgac
     2341 agctttgatg agatgaatgc agagctgcag tcaaaactga aggatttatt taatgtggat
     2401 gcttttaagc tggaatcatt agaagcaaaa aacagagcat tgaatgaaca gattgcaaga
     2461 ttggaacaag aaagagaaaa agaaccgaat cgtctagagt cgttgagaaa actgaaggct
     2521 tccttacaag gagatgttca aaagtatcag gcatacatga gcaatttgga gtctcattca
     2581 gccattcttg accagaaatt aaatggtctc aatgaggaaa ttgctagagt agaactagaa
     2641 tgtgaaacaa taaaacagga gaacactcga ctacagaata tcattgacaa ccagaagtac
     2701 tcagttgcag acattgagcg aataaatcat gaaagaaatg aattgcagca gactattaat
     2761 aaattaacca aggacctgga agctgaacaa cagaagttgt ggaatgagga gttaaaatat
     2821 gccagaggca aagaagcgat tgaaacacaa ttagcagagt atcacaaatt ggctagaaaa
     2881 ttaaaactta ttcctaaagg tgctgagaat tccaaaggtt atgactttga aattaagttt
     2941 aatcccgagg ctggtgccaa ctgccttgtc aaatacaggg ctcaagttta tgtacctctt
     3001 aaggaactcc tgaatgaaac tgaagaagaa attaataaag ccctaaataa aaaaatgggt
     3061 ttggaggata ctttagaaca attgaatgca atgataacag aaagcaagag aagtgtgaga
     3121 actctgaaag aagaagttca aaaattggat gatctttacc aacaaaaaat taaggaagca
     3181 gaggaagagg atgaaaaatg tgccagtgag cttgagtcct tggagaaaca caagcacctg
     3241 ctagaaagta ctgttaacca ggggctcagt gaagctatga atgaattaga tgctgttcag
     3301 cgggaatacc aactagttgt gcaaaccacg actgaagaaa gacgaaaagt gggaaataac
     3361 ttgcaacgtc tgttagagat ggttgctaca catgttgggt ctgtagagaa acatcttgag
     3421 gagcagattg ctaaagttga tagagaatat gaagaatgca tgtcagaaga tctctcggaa
     3481 aatattaaag agattagaga taagtatgag aagaaagcta ctctaattaa gtcttctgaa
     3541 gaatgactcg acggtaccgc gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     3601 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3661 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3721 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3781 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3841 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3901 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3961 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4021 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     4081 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     4141 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtg ctggcgcgcc accgagtaca
     4201 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     4261 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc
     4321 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     4381 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     4441 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     4501 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     4561 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     4621 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     4681 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     4741 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacct aggcacgtac
     4801 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4861 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4921 ccaacttgtt tattgcagct taaatggtta caaataaagc aatagcatca caaatttcac
     4981 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5041 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5101 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5161 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5221 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5281 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5341 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5401 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5461 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5521 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5581 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5641 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5701 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5761 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5821 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5881 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5941 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6001 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6061 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6121 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6181 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6241 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6301 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6361 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6421 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6481 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6541 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6601 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6661 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6721 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6781 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6841 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6901 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6961 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7021 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7081 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7141 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7201 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7261 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7321 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     7381 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     7441 cgt
//
