LOCUS       sequence-416605-        6329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of N-terminally EGFP-tagged NIP45 delta-N2 
            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 6329)
  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 6329)
  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 delta-N2
FEATURES             Location/Qualifiers
     source          1..6329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        123..502
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        503..706
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     657..677
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    708..726
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    729..747
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     757..781
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(877..879,880..882,883..1593)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(877..879,880..882,883..1593)
                     /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(922..943)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1183..1202)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1530..1551
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2169..2186)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2175..2399
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2532..2551)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2683..2730
                     /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             2738..3760
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2738..3760
                     /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    3890..4011
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3927..3946)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3981..4000
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4060..4076)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4060..4076)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4073..4095)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4084..4100
                     /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(4108..4114,4115..4132,4133..4138))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4153..4174
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4291..4308)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4462..5050)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4542..4561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5221..6012,6013..6081))
                     /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     5844..5863
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6082..6186)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6261..6280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
       61 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      121 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      181 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      241 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      301 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      361 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      421 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      481 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      541 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      601 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      661 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctcc ctatcagtga
      721 tagagatctc cctatcagtg atagagatcg tcgacgagct cgtttagtga accgtcagat
      781 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
      841 cctccggact ctagcgttta aacttaagct tccaccatgg tgagcaaggg cgaggagctg
      901 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      961 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1021 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     1081 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1141 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1201 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1261 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1321 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     1381 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1441 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1501 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1561 gggatcactc tcggcatgga cgagctgtac aagaagcttg gtaccggcga agggcaaggg
     1621 caagggcaag ggccgggccg cggcaccccc cgactcttcc cactcaaaat ccgttgccgg
     1681 gctgacctgg tcagattgcc cctcaggatg tcggagcccc tgcagagtgt ggtggaccac
     1741 atggccaccc accttggggt gtccccaagc aggatccttt tgctttttgg agagacagag
     1801 ctatcaccta ctgccactcc caggacccta aagctcggag tggctgacat cattgactgt
     1861 gtggtactaa caagttctcc agaggccaca gagacgtccc aacagctcca gctccgggtg
     1921 cagggaaagg agaaacacca gacactggaa gtctcactgt ctcgagattc ccctctaaag
     1981 accctcatgt cccactatga ggaggccatg ggactgtcgg gacggaagct ctccttcttc
     2041 tttgatggga caaagctttc aggcagggag ctgccagctg acctgggcat ggaatctggg
     2101 gacctcattg aggtctgggg ctgagcggcc gctcgagtct agagggcccg tttaaacccg
     2161 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2221 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2281 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2341 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2401 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2461 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2521 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2581 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2641 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     2701 ttcctattct ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac
     2761 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc
     2821 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     2881 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     2941 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta
     3001 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3061 cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3121 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3181 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3241 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3301 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     3361 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     3421 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     3481 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     3541 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     3601 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     3661 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     3721 aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga
     3781 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3841 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     3901 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3961 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4021 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4081 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4141 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4201 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4261 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4321 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4381 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4441 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4501 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4561 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4621 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4681 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4741 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4801 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4861 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4921 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4981 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5041 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5101 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5161 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5221 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5281 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5341 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5401 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5461 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5521 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5581 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5641 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5701 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5761 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5821 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5881 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5941 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6001 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6061 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6121 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6181 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     6241 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     6301 ctccctgctt gtgtgttgga ggtcgctga
//
