LOCUS       sequence-221115-        7588 bp    DNA     circular SYN 15-APR-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 7588)
  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 7588)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-d80
FEATURES             Location/Qualifiers
     source          1..7588
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        55..434
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        435..638
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     589..609
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    640..658
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    661..679
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     689..713
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      894..903
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(900..902,903..905,906..1616)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(900..902,903..905,906..1616)
                     /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(945..966)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1206..1225)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1553..1574
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3360..3377)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3366..3590
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3723..3742)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3874..3921
                     /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             3929..4951
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3929..4951
                     /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    5081..5202
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5118..5137)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5172..5191
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5251..5267)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5251..5267)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5264..5286)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5275..5291
                     /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(5299..5305,5306..5323,5324..5329))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5344..5365
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5482..5499)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5653..6241)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5733..5752)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6412..7203,7204..7272))
                     /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     7035..7054
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7273..7377)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7452..7471)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
       61 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      121 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      181 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      241 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      301 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      361 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      421 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      481 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      541 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      601 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      661 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      721 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      781 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
      841 attctgcaga tatccagcac agtggcggcc gctcgagtct agcgctaccg gtcgccacca
      901 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
      961 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     1021 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     1081 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     1141 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     1201 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     1261 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1321 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     1381 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1441 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1501 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1561 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtccg
     1621 gactcagatc caaggacccg agaccactta acgacaaagc attcattcag cagtgtattc
     1681 gacaactctg tgagtttctt acagaaaatg gttatgcaca taatgtgtcc atgaaatctc
     1741 tacaagctcc ctctgttaaa gacttcctga agatcttcac atttctttat ggcttcctgt
     1801 gcccctcata cgaacttcct gacacaaagt ttgaagaaga ggttccaaga atctttaaag
     1861 accttgggta tccttttgca ctatccaaaa gctccatgta cacagtgggg gctcctcata
     1921 catggcctca cattgtggca gccttagttt ggctaataga ctgcatcaag atacatactg
     1981 ccatgaaaga aagctcacct ttatttgatg atgggcagcc ttggggagaa gaaactgaag
     2041 atggaattat gcataataag ttgtttttgg actacaccat aaaatgctat gagagtttta
     2101 tgagtggtgc cgacagcttt gatgagatga atgcagagct gcagtcaaaa ctgaaggatt
     2161 tatttaatgt ggatgctttt aagctggaat cattagaagc aaaaaacaga gcattgaatg
     2221 aacagattgc aagattggaa caagaaagag aaaaagaacc gaatcgtcta gagtcgttga
     2281 gaaaactgaa ggcttcctta caaggagatg ttcaaaagta tcaggcatac atgagcaatt
     2341 tggagtctca ttcagccatt cttgaccaga aattaaatgg tctcaatgag gaaattgcta
     2401 gagtagaact agaatgtgaa acaataaaac aggagaacac tcgactacag aatatcattg
     2461 acaaccagaa gtactcagtt gcagacattg agcgaataaa tcatgaaaga aatgaattgc
     2521 agcagactat taataaatta accaaggacc tggaagctga acaacagaag ttgtggaatg
     2581 aggagttaaa atatgccaga ggcaaagaag cgattgaaac acaattagca gagtatcaca
     2641 aattggctag aaaattaaaa cttattccta aaggtgctga gaattccaaa ggttatgact
     2701 ttgaaattaa gtttaatccc gaggctggtg ccaactgcct tgtcaaatac agggctcaag
     2761 tttatgtacc tcttaaggaa ctcctgaatg aaactgaaga agaaattaat aaagccctaa
     2821 ataaaaaaat gggtttggag gatactttag aacaattgaa tgcaatgata acagaaagca
     2881 agagaagtgt gagaactctg aaagaagaag ttcaaaagct ggatgatctt taccaacaaa
     2941 aaattaagga agcagaggaa gaggatgaaa aatgtgccag tgagcttgag tccttggaga
     3001 aacacaagca cctgctagaa agtactgtta accaggggct cagtgaagct atgaatgaat
     3061 tagatgctgt tcagcgggaa taccaactag ttgtgcaaac cacgactgaa gaaagacgaa
     3121 aagtgggaaa taacttgcaa cgtctgttag agatggttgc tacacatgtt gggtctgtag
     3181 agaaacatct tgaggagcag attgctaaag ttgatagaga atatgaagaa tgcatgtcag
     3241 aagatctctc ggaaaatatt aaagagatta gagataagta tgagaagaaa gctactctaa
     3301 ttaagtcttc tgaagaatga ctcgacggta ccgcgggccc gtttaaaccc gctgatcagc
     3361 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3421 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3481 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3541 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     3601 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3661 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3721 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3781 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3841 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     3901 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     3961 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     4021 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     4081 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     4141 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     4201 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     4261 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     4321 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     4381 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     4441 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     4501 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     4561 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     4621 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     4681 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     4741 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     4801 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     4861 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     4921 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     4981 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5041 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5101 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5161 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5221 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5281 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5341 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5401 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5461 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5521 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5581 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5641 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5701 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5761 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5821 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5881 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5941 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6001 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6061 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6121 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6181 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6241 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6301 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6361 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6421 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6481 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6541 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6601 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6661 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6721 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6781 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6841 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6901 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6961 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7021 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7081 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7141 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7201 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7261 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7321 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7381 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7441 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     7501 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     7561 ttgaccgaca attgcatgaa gaatctgc
//
