LOCUS       sequence-416599-        7070 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of N-terminally EGFP-tagged NIP45 wt 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 wt
FEATURES             Location/Qualifiers
     source          1..7070
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..66
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     306..327
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(468..492)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    502..520
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    523..541
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(543..746)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(572..592)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        747..1126
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1298..1317
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1392..1496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1497..1565,1566..2357)
                     /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     complement(1715..1734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2528..3116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3017..3036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3270..3287
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3404..3425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3440..3445,3446..3463,3464..3470)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3478..3494
                     /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)."
     primer_bind     3483..3505
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3502..3518
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3502..3518
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3567..3688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3578..3597)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3632..3651
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3818..4840)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             complement(3818..4840)
                     /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
                     "
     protein_bind    complement(4848..4895)
                     /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)."
     primer_bind     5027..5046
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5179..5403
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5392..5409
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(6726..7070,1..366,367..369,370..372))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(6726..7070,1..366,367..369,370..372))
                     /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(6768..6789)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
       61 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
      121 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag
      181 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
      241 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
      301 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
      361 cttgctcacc atggtggaag cttaagttta aacgctagag tccggaggct ggatcggtcc
      421 cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc
      481 actaaacgag ctcgtcgacg atctctatca ctgataggga gatctctatc actgataggg
      541 agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg
      601 gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca
      661 ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca
      721 ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac
      781 tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta
      841 ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag
      901 tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt
      961 actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca
     1021 ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat
     1081 gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg
     1141 gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg
     1201 tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc
     1261 aacacacaag cagggagcag atactggctt aactatgcgg catcagagca gattgtactg
     1321 agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc
     1381 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1441 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1501 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1561 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1621 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1681 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1741 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1801 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1861 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1921 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1981 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2041 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2101 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2161 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2221 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2281 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2341 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2401 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2461 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2521 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2581 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2641 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     2701 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2761 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2821 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2881 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2941 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3001 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3061 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3121 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3181 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3241 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3301 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     3361 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     3421 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     3481 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc
     3541 tagaggtcga cggtatacag acatgataag atacattgat gagtttggac aaaccacaac
     3601 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3661 aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc atgagatccc
     3721 cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat
     3781 agaaggcggc ggtggaatcg aaatctcgta gtacgtgcta ttcctttgcc ctcggacgag
     3841 tgctggggcg tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg
     3901 ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg
     3961 cgtcgcatcg accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat
     4021 agagttggtc aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct
     4081 ccggatgcct ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc
     4141 agaagaagat gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa
     4201 tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca
     4261 cgaggtgccg gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg
     4321 cgacggacgc actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag
     4381 caatcgcgca tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg
     4441 ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga
     4501 ccggctgcag aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg
     4561 cacggcggga gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg
     4621 gaatcgggag cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat
     4681 cggcgcagct atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac
     4741 gagattcttc gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca
     4801 gaaacttctc gacagacgtc gcggtgagtt caggcttttt ggccaaggaa gttcctatac
     4861 tttctagaga ataggaactt cggaatagga acttctaggt acgtgaacca tcaccctaat
     4921 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     4981 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5041 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5101 ccgccgcgct taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg
     5161 ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt
     5221 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
     5281 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
     5341 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
     5401 cagtcgaggc tgatcagcgg gtttaaacgg gccctctaga ctcgagcggc cgctcagccc
     5461 cagacctcaa tgaggtcccc agattccatg cccaggtcag ctggcagctc cctgcctgaa
     5521 agctttgtcc catcaaagaa gaaggagagc ttccgtcccg acagtcccat ggcctcctca
     5581 tagtgggaca tgagggtctt tagaggggaa tctcgagaca gtgagacttc cagtgtctgg
     5641 tgtttctcct ttccctgcac ccggagctgg agctgttggg acgtctctgt ggcctctgga
     5701 gaacttgtta gtaccacaca gtcaatgatg tcagccactc cgagctttag ggtcctggga
     5761 gtggcagtag gtgatagctc tgtctctcca aaaagcaaaa ggatcctgct tggggacacc
     5821 ccaaggtggg tggccatgtg gtccaccaca ctctgcaggg gctccgacat cctgaggggc
     5881 aatctgacca ggtcagcccg gcaacggatt ttgagtggga agagtcgggg ggtctctggg
     5941 agggtgggcc cttccaccaa gaccacttca tcctcttggc cctgttgctc ttgaccctga
     6001 ggtggcttgg ggctcagaca ggaacggaga tcctggaggc gcttgttcac ctcacttaac
     6061 ttcttgagtg cccgagtatg ggttcgagac ttcgttcttg gtgaaggtgg ggacagagga
     6121 gagttgtcca gatccagaaa ctctgtcttt ttcttctcct ccttgtcttt tgttcgaagc
     6181 tttgtcttcc agggagagcc tggtgatggg gagccctcat ggtagagacc actcgaatct
     6241 gccagctctg cctcttcctc atcccctgga gggtagagtt tcaggaggga tagatcatca
     6301 ggaatgagac ggagactgct tttaaccttc cccgagtaca ccggaaccag cggcgcctcc
     6361 cccggatcca gcaccagccg ccgccgccgc ctgaccggct cccgcggggg tcctgcgggc
     6421 cgcctgtcct ccccttcgct gtcactgttg ctgttatccc gggacgcgac cggccccggg
     6481 ggctccgggg gctccacctc aacctcgtcc gcggcaccgc gagcggtggc gacctccaga
     6541 atttcctcat cgctgtcggt gaccaagtcc acagacacta cgtccagcgt gccccgggat
     6601 ggagaccgct gggcccgagg acgccggccc cgaccgcccc agccgccccg accccctcgg
     6661 ccggcaccgc tacctccgga ccagcggccc cgcttcccca caggctccgc catggtacca
     6721 agcttcttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     6781 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     6841 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     6901 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     6961 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     7021 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
//
