LOCUS       sequence-215367-        7211 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 7211)
  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 7211)
  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..7211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1074..1083
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1080..1082,1083..1085,1086..1796)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1080..1082,1083..1085,1086..1796)
                     /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(1125..1146)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1386..1405)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1733..1754
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3549..3566)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3555..3779
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3912..3931)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4062..4109
                     /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             4153..4749
                     /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     4646..4666
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4885..5005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4921..4940)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4975..4994
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5054..5070)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5054..5070)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5067..5089)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5078..5094
                     /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(5102..5108,5109..5126,5127..5132))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5147..5168
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5285..5302)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5456..6044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5536..5555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6215..7006,7007..7075))
                     /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     6838..6857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7076..7180)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatccagcac agtggcggcc gctcgagtct agcgctaccg gtcgccacca
     1081 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     1141 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1201 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1261 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1321 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1381 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1441 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1501 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1561 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1621 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1681 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1741 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg
     1801 gactcagatc catgatcaag gacccgagac cacttaacga caaagcattc attcagcagt
     1861 gtattcgaca actctgtgag tttcttacag aaaatggtta tgcacataat gtgtccatga
     1921 aatctctaca agctccctct gttaaagact tcctgaagat cttcacattt ctttatggct
     1981 tcctgtgccc ctcatacgaa cttcctgaca caaagtttga agaagaggtt ccaagaatct
     2041 ttaaagacct tgggtatcct tttgcactat ccaaaagctc catgtacaca gtgggggctc
     2101 ctcatacatg gcctcacatt gtggcagcct tagtttggct aatagactgc atcaagatac
     2161 atactgccat gaaagaaagc tcacctttat ttgatgatgg gcagccttgg ggagaagaaa
     2221 ctgaagatgg aattatgcat aataagttgt ttttggacta caccataaaa tgctatgaga
     2281 gttttatgag tggtgccgac agctttgatg agatgaatgc agagctgcag tcaaaactga
     2341 aggatttatt taatgtggat gcttttaagc tggaatcatt agaagcaaaa aacagagcat
     2401 tgaatgaaca gattgcaaga ttggaacaag aaagagaaaa agaaccgaat cgtctagagt
     2461 cgttgagaaa actgaaggct tccttacaag gagatgttca aaagtatcag gcatacatga
     2521 gcaatttgga gtctcattca gccattcttg accagaaatt aaatggtctc aatgaggaaa
     2581 ttgctagagt agaactagaa tgtgaaacaa taaaacagga gaacactcga ctacagaata
     2641 tcattgacaa ccagaagtac tcagttgcag acattgagcg aataaatcat gaaagaaatg
     2701 aattgcagca gactattaat aaattaacca aggacctgga agctgaacaa cagaagttgt
     2761 ggaatgagga gttaaaatat gccagaggca aagaagcgat tgaaacacaa ttagcagagt
     2821 atcacaaatt ggctagaaaa ttaaaactta ttcctaaagg tgctgagaat tccaaaggtt
     2881 atgactttga aattaagttt aatcccgagg ctggtgccaa ctgccttgtc aaatacaggg
     2941 ctcaagttta tgtacctctt aaggaactcc tgaatgaaac tgaagaagaa attaataaag
     3001 ccctaaataa aaaaatgggt ttggaggata ctttagaaca attgaatgca atgataacag
     3061 aaagcaagag aagtgtgaga actctgaaag aagaagttca aaaattggat gatctttacc
     3121 aacaaaaaat taaggaagca gaggaagagg atgaaaaatg tgccagtgag cttgagtcct
     3181 tggagaaaca caagcacctg ctagaaagta ctgttaacca ggggctcagt gaagctatga
     3241 atgaattaga tgctgttcag cgggaatacc aactagttgt gcaaaccacg actgaagaaa
     3301 gacgaaaagt gggaaataac ttgcaacgtc tgttagagat ggttgctaca catgttgggt
     3361 ctgtagagaa acatcttgag gagcagattg ctaaagttga tagagaatat gaagaatgca
     3421 tgtcagaaga tctctcggaa aatattaaag agattagaga taagtatgag aagaaagcta
     3481 ctctaattaa gtcttctgaa gaatgactcg aggacggtac cgcgggcccg tttaaacccg
     3541 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3601 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3661 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3721 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3781 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3841 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3901 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3961 ccgtcaagct ctaaatcggg ggtcccttta gggttccgat ttagtgcttt acggcacctc
     4021 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     4081 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     4141 tgctggcgcg ccaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     4201 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     4261 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     4321 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     4381 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     4441 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     4501 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     4561 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     4621 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     4681 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     4741 ggtgcctgac ctaggcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4801 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4861 atgctggagt tcttcgccca ccccaacttg tttattgcag cttaaatggt tacaaataaa
     4921 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4981 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5041 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5101 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5161 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5221 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5281 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5341 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5401 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5461 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5521 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5581 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5641 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5701 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5761 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5821 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5881 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5941 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6001 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6061 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6121 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6181 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6241 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6301 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6361 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6421 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6481 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6541 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6601 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6661 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6721 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6781 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6841 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6901 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6961 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7021 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7081 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7141 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7201 cacctgacgt c
//
