LOCUS       sequence-272295-        7227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of Flip in SKAP and IRES GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7227)
  AUTHORS   Kern DM, Nicholls PK, Page DC, Cheeseman IM
  TITLE     A mitotic SKAP isoform regulates spindle positioning at astral 
            microtubule plus ends.
  JOURNAL   J Cell Biol. 2016 May 9;213(3):315-28. doi: 10.1083/jcb.201510117. 
            Epub 2016 May 2.
  PUBMED    27138257
REFERENCE   2  (bases 1 to 7227)
  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"; doi: 
            "10.1083/jcb.201510117"; journalName: "J Cell Biol"; date: 
            "2016-05-9- 9"; volume: "213"; issue: "3"; pages: "315-28"
COMMENT     Sequence Label: pDK480
FEATURES             Location/Qualifiers
     source          1..7227
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        101..480
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        481..684
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     635..655
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    686..704
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    707..725
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     735..759
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     misc_feature    join(1604..2177,2178..2180,2181..2189)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1770..1787)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1997..2016
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2190..2192,2193..2195,2196..2909)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2190..2192,2193..2195,2196..2906)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2235..2256)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2496..2515)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2843..2864
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3045..3062)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3051..3275
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3408..3427)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3559..3606
                     /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             3614..4636
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4766..4887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4803..4822)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4857..4876
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4936..4952)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4936..4952)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4949..4971)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4960..4976
                     /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(4984..4990,4991..5008,5009..5014))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5029..5050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5167..5184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5338..5926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5418..5437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6097..6888,6889..6957))
                     /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     6720..6739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6958..7062)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7137..7156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
       61 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      121 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      181 acataactta cggtaaatgg cccgcctggc tgaccgccca atgacccccg cccattgacg
      241 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      301 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      361 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      421 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      481 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      541 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      601 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      661 tgggaggtct atataagcag agctctccct atcagtgata gagatctccc tatcagtgat
      721 agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc
      781 tgttttgacc tccatagaag acaccgggac cgatccaacc tccggactct agcgtttgct
      841 accggtcctc gaggccacca tgaccagtgt ggttaagaca gtgtatagcc tgcagccccc
      901 ctctgcgctg agcggcggcc agccggcaga cacacaaact cgggccactt ctaagagtct
      961 cttacctgtt aggtccaaag aagtcgatgt ttccaaacag cttcattcag gaggtccaga
     1021 gaatgatgtt acaaaaatca ccaaactgag acgagagaat gggcaaatga aagctactga
     1081 cactgccacc agaaggaatg tcagaaaggg atataagcct ttatccaagc aaaaatcaga
     1141 ggaagagctc aaggacaaga accagctgtt agaagccgtc aacaagcagt tgcaccagaa
     1201 gttgactgaa actcagggag agctgaagga cctgacccag aaggtagagc tgctggagaa
     1261 gtttcgggac aactgtttgg caattttgga gagcaagggc cttgatccag ctttaggcag
     1321 tgagaccctg gcatcacgac aagaatccac tactgatcac atggactcta tgttgctgtt
     1381 agaaactttg caagaggagc tgaagctttt taacgaaaca gccaaaaagc agatggagga
     1441 gttacaggcc ttaaaggtaa agctggagat gaaagaggag cgcgtgaggt ttttggaaca
     1501 gcaaacctta tgtaacaatc aagtaaatga tttaacaaca gcccttaagg aaatggagca
     1561 gctattagaa atgtaagaat tctgcagtcg acggtaccgc gggcccctct ccctcccccc
     1621 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     1681 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     1741 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     1801 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     1861 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1921 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1981 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     2041 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt
     2101 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     2161 aaaaacacga tgataatatg gccacaacca tggtgagcaa gggcgaggag ctgttcaccg
     2221 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     2281 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     2341 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     2401 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     2461 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     2521 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     2581 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     2641 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     2701 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     2761 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     2821 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     2881 ctctcggcat ggacgagctg tacaagtaag gatccaccgg atctagataa ctgatcataa
     2941 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3001 tgaacctgaa acataaaatg aatgcaatta aacccgctga tcagcctcga ctgtgccttc
     3061 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3121 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3181 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3241 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3301 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3361 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3421 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3481 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3541 tgatggttca cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg
     3601 aacttccttg gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     3661 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     3721 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     3781 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     3841 gcttgacatt ggggagttca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     3901 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga
     3961 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     4021 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
     4081 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     4141 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     4201 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     4261 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     4321 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     4381 aggatcgcca cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     4441 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     4501 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     4561 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     4621 gagggcaaag gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag
     4681 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcagggatct
     4741 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4801 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4861 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4921 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4981 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5041 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5101 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5161 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5221 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5281 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5341 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5401 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5461 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5521 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5581 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5641 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5701 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5761 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5821 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5881 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5941 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6001 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6061 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6181 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6241 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6301 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6361 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6421 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6481 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6541 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6601 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6661 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6721 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6781 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6841 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6901 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6961 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7021 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7081 gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca
     7141 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc
     7201 gctgagtagt gcgcgagcaa aatttaa
//
