LOCUS       sequence-416603-        7070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of N-terminally EGFP-tagged NIP45 D394R 
            resistant to siRNA. Use with Flp-In T-Rex system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7070)
  AUTHORS   Hertz EPT, Vega IA, Kruse T, Wang Y, Hendriks IA, Bizard AH, 
            Eugui-Anta A, Hay RT, Nielsen ML, Nilsson J, Hickson ID, Mailand N
  TITLE     The SUMO-NIP45 pathway processes toxic DNA catenanes to prevent 
            mitotic failure.
  JOURNAL   Nat Struct Mol Biol. 2023 Jul 20. doi: 10.1038/s41594-023-01045-0.
  PUBMED    37474739
REFERENCE   2  (bases 1 to 7070)
  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 Struct 
            Mol Biol. 2023 Jul 20. doi: 10.1038/s41594-023-01045-0."
COMMENT     Sequence Label: EGFP-NIP45 D394R
FEATURES             Location/Qualifiers
     source          1..7070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(217..236)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     564..585
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1944..1961)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1950..2174
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2307..2326)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2458..2505
                     /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             2513..3535
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2513..3535
                     /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    3665..3786
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3702..3721)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3756..3775
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3835..3851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3835..3851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3848..3870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3859..3875
                     /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(3883..3889,3890..3907,3908..3913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3928..3949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4066..4083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4237..4825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4317..4336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4996..5787,5788..5856))
                     /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     5619..5638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5857..5961)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6036..6055)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6227..6606
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6607..6810
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6761..6781
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6812..6830
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6833..6851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6861..6885
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(6981..6983,6984..6986,6987..7070,1..627)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6981..6983,6984..6986,6987..7070,1..627)
                     /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(7026..7047)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
       61 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      121 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      181 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      241 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      301 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      361 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
      421 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      481 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
      541 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
      601 actctcggca tggacgagct gtacaagaag cttggtacca tggcggagcc tgtggggaag
      661 cggggccgct ggtccggagg tagcggtgcc ggccgagggg gtcggggcgg ctggggcggt
      721 cggggccggc gtcctcgggc ccagcggtct ccatcccggg gcacgctgga cgtagtgtct
      781 gtggacttgg tcaccgacag cgatgaggaa attctggagg tcgccaccgc tcgcggtgcc
      841 gcggacgagg ttgaggtgga gcccccggag cccccggggc cggtcgcgtc ccgggataac
      901 agcaacagtg acagcgaagg ggaggacagg cggcccgcag gacccccgcg ggagccggtc
      961 aggcggcggc ggcggctggt gctggatccg ggggaggcgc cgctggttcc ggtgtactcg
     1021 gggaaggtta aaagcagttt gcgtttgatc ccagatgatc tatccctcct gaaactctac
     1081 cctccagggg atgaggaaga ggcagagctg gcagattcga gtggtctcta ccatgagggc
     1141 tccccatcac caggctctcc ctggaagaca aagctgagga ctaaggataa agaagagaag
     1201 aaaaagacag agtttctgga tctggacaac tctcctctgt ccccaccttc accaaggacc
     1261 aaaagcagaa cgcatactcg ggcactcaag aagttaagtg aggtgaacaa gcgcctccag
     1321 gatctccgtt cctgtctgag ccccaagcca cctcagggtc aagagcaaca gggccaagag
     1381 gatgaagtgg tcttggtgga agggcccacc ctcccagaga ccccccgact cttcccactc
     1441 aaaatccgtt gccgggctga cctggtcaga ttgcccctca ggatgtcgga gcccctgcag
     1501 agtgtggtgg accacatggc cacccacctt ggggtgtccc caagcaggat ccttttgctt
     1561 tttggagaga cagagctatc acctactgcc actcccagga ccctaaagct cggagtggct
     1621 gacatcattg actgtgtggt actaacaagt tctccagagg ccacagagac gtcccaacag
     1681 ctccagctcc gggtgcaggg aaaggagaaa caccagacac tggaagtctc actgtctcga
     1741 gattcccctc taaagaccct catgtcccac tatgaggagg ccatgggact gtcgggacgg
     1801 aagctctcct tcttctttcg tgggacaaag ctttcaggca gggagctgcc agctgacctg
     1861 ggcatggaat ctggggacct cattgaggtc tggggctgag cggccgctcg agtctagagg
     1921 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     1981 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2041 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2101 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2161 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2221 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2281 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2341 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2401 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     2461 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     2521 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     2581 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     2641 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     2701 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     2761 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     2821 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     2881 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     2941 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     3001 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     3061 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     3121 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     3181 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     3241 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     3301 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     3361 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     3421 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     3481 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     3541 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3601 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3661 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3721 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3781 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3841 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3901 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3961 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4021 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4081 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4141 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4201 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4261 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4321 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4381 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4441 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4501 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4561 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4621 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4681 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4741 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4801 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4861 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4921 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4981 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5041 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5101 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5161 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5221 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5281 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5341 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5401 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5461 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5521 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5581 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5641 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5701 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5761 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5821 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5881 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5941 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     6001 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     6061 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     6121 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     6181 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     6241 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     6301 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     6361 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     6421 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     6481 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     6541 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     6601 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     6661 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     6721 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     6781 gtacggtggg aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc
     6841 agtgatagag atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
     6901 ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg
     6961 tttaaactta agcttccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     7021 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
//
