LOCUS       sequence-220985-        7443 bp    DNA     circular SYN 14-APR-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 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 Apr 14, 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
FEATURES             Location/Qualifiers
     source          1..7443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        91..470
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        471..674
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     625..645
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    676..694
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    697..715
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     725..749
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      930..939
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(936..938,939..941,942..1652)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(936..938,939..941,942..1652)
                     /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(981..1002)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1242..1261)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1589..1610
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3636..3653)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3642..3866
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3999..4018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4150..4197
                     /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             4241..4837
                     /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     4734..4754
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4973..5093
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5009..5028)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5063..5082
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5142..5158)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5142..5158)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5155..5177)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5166..5182
                     /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(5190..5196,5197..5214,5215..5220))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5235..5256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5373..5390)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5544..6132)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5624..5643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6303..7094,7095..7163))
                     /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     6926..6945
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7164..7268)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7343..7362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
       61 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
      121 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      181 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      241 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      301 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      361 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      421 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      481 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      541 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      601 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      661 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      721 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      781 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttg
      841 gtaccgagct cggatccact agtccagtgt ggtggaattc tgcagatatc cagcacagtg
      901 gcggccgctc gagtctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt
      961 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1021 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1081 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1141 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1201 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1261 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1321 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1381 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1441 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1501 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1561 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1621 ggatcactct cggcatggac gagctgtaca agtccggact cagatccatg aagcgcagtt
     1681 cagtttccag cggtggtgct ggccgcctct ccatgcagga gttaagatcc caggatgtaa
     1741 ataaacaagg cctctatacc cctcaaacca aagagaaacc aacctttgga aagttgagta
     1801 taaacaaacc gacatctgaa agaaaagtct cgctatttgg caaaagaact agtggacatg
     1861 gatcacggaa tagtcaactt ggtatatttt ccagttctga gaaaatcaag gacccgagac
     1921 cacttaacga caaagcattc attcagcagt gtattcgaca actctgtgag tttcttacag
     1981 aaaatggtta tgcacataat gtgtccatga aatctctaca agctccctct gttaaagact
     2041 tcctgaagat cttcacattt ctttatggct tcctgtgccc ctcatacgaa cttcctgaca
     2101 caaagtttga agaagaggtt ccaagaatct ttaaagacct tgggtatcct tttgcactat
     2161 ccaaaagctc catgtacaca gtgggggctc ctcatacatg gcctcacatt gtggcagcct
     2221 tagtttggct aatagactgc atcaagatac atactgccat gaaagaaagc tcacctttat
     2281 ttgatgatgg gcagccttgg ggagaagaaa ctgaagatgg aattatgcat aataagttgt
     2341 ttttggacta caccataaaa tgctatgaga gttttatgag tggtgccgac agctttgatg
     2401 agatgaatgc agagctgcag tcaaaactga aggatttatt taatgtggat gcttttaagc
     2461 tggaatcatt agaagcaaaa aacagagcat tgaatgaaca gattgcaaga ttggaacaag
     2521 aaagagaaaa agaaccgaat cgtctagagt cgttgagaaa actgaaggct tccttacaag
     2581 gagatgttca aaagtatcag gcatacatga gcaatttgga gtctcattca gccattcttg
     2641 accagaaatt aaatggtctc aatgaggaaa ttgctagagt agaactagaa tgtgaaacaa
     2701 taaaacagga gaacactcga ctacagaata tcattgacaa ccagaagtac tcagttgcag
     2761 acattgagcg aataaatcat gaaagaaatg aattgcagca gactattaat aaattaacca
     2821 aggacctgga agctgaacaa cagaagttgt ggaatgagga gttaaaatat gccagaggca
     2881 aagaagcgat tgaaacacaa ttagcagagt atcacaaatt ggctagaaaa ttaaaactta
     2941 ttcctaaagg tgctgagaat tccaaaggtt atgactttga aattaagttt aatcccgagg
     3001 ctggtgccaa ctgccttgtc aaatacaggg ctcaagttta tgtacctctt aaggaactcc
     3061 tgaatgaaac tgaagaagaa attaataaag ccctaaataa aaaaatgggt ttggaggata
     3121 ctttagaaca attgaatgca atgataacag aaagcaagag aagtgtgaga actctgaaag
     3181 aagaagttca aaagctggat gatctttacc aacaaaaaat taaggaagca gaggaagagg
     3241 atgaaaaatg tgccagtgag cttgagtcct tggagaaaca caagcacctg ctagaaagta
     3301 ctgttaacca ggggctcagt gaagctatga atgaattaga tgctgttcag cgggaatacc
     3361 aactagttgt gcaaaccacg actgaagaaa gacgaaaagt gggaaataac ttgcaacgtc
     3421 tgttagagat ggttgctaca catgttgggt ctgtagagaa acatcttgag gagcagattg
     3481 ctaaagttga tagagaatat gaagaatgca tgtcagaaga tctctcggaa aatattaaag
     3541 agattagaga taagtatgag aagaaagcta ctctaattaa gtcttctgaa gaatgactcg
     3601 acggtaccgc gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3661 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3721 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3781 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3841 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3901 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3961 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4021 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4081 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4141 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     4201 ggccaaaaag cctgaactca ccgcgacgtg ctggcgcgcc accgagtaca agcccacggt
     4261 gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc
     4321 cgactacccc gccacgcgcc acaccgtcga cccggaccgc cacatcgagc gggtcaccga
     4381 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     4441 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     4501 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     4561 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     4621 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     4681 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     4741 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     4801 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacct aggcacgtac tacgagattt
     4861 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4921 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4981 tattgcagct taaatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5041 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5101 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5161 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5221 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5281 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5341 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5401 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5461 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5521 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5581 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5641 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5701 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5761 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5821 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5881 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5941 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6001 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6061 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6121 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6181 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6241 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6301 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6361 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6421 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6481 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6541 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6601 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6661 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6721 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6781 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6841 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6901 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6961 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7021 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7081 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7141 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7201 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7261 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     7321 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     7381 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     7441 caa
//
