LOCUS       sequence-416604-        7052 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible expression of N-terminally mCherry-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 7052)
  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 7052)
  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 Struct 
            Mol Biol. 2023 Jul 20. doi: 10.1038/s41594-023-01045-0."
COMMENT     Sequence Label: mCherry-NIP45 D394R
FEATURES             Location/Qualifiers
     source          1..7052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..32)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     67..86
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(146..162)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(146..162)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(159..181)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    170..186
                     /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(194..200,201..218,219..224))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    239..260
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(377..394)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(548..1136)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(628..647)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1307..2098,2099..2167))
                     /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     1930..1949
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2168..2272)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2347..2366)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2538..2917
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2918..3121
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3072..3092
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    3123..3141
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3144..3162
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3172..3196
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3283..3292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3289..3996
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3434..3452)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3702..3721
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(5307..5324)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5313..5537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5670..5689)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5821..5868
                     /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             5876..6898
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5876..6898
                     /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    join(7028..7052,1..97)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
       61 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
      121 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
      181 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
      241 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
      301 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      361 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
      421 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
      481 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
      541 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
      601 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
      661 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
      721 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
      781 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
      841 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
      901 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
      961 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1021 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1081 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1141 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1201 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1261 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1321 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1381 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1441 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1501 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     1561 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     1621 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     1681 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     1741 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     1801 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     1861 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     1921 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     1981 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2041 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2101 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2161 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2221 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     2281 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     2341 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     2401 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     2461 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     2521 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     2581 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     2641 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     2701 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     2761 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     2821 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     2881 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     2941 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     3001 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     3061 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     3121 tctccctatc agtgatagag atctccctat cagtgataga gatcgtcgac gagctcgttt
     3181 agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca
     3241 ccgggaccga tccagcctcc ggactctagc gtttaaactt aagccaccat ggtgagcaag
     3301 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     3361 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     3421 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     3481 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     3541 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     3601 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     3661 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     3721 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     3781 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     3841 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     3901 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     3961 cgccactcca ccggcggcat ggacgagctg tacaagggta ccatggcgga gcctgtgggg
     4021 aagcggggcc gctggtccgg aggtagcggt gccggccgag ggggtcgggg cggctggggc
     4081 ggtcggggcc ggcgtcctcg ggcccagcgg tctccatccc ggggcacgct ggacgtagtg
     4141 tctgtggact tggtcaccga cagcgatgag gaaattctgg aggtcgccac cgctcgcggt
     4201 gccgcggacg aggttgaggt ggagcccccg gagcccccgg ggccggtcgc gtcccgggat
     4261 aacagcaaca gtgacagcga aggggaggac aggcggcccg caggaccccc gcgggagccg
     4321 gtcaggcggc ggcggcggct ggtgctggat ccgggggagg cgccgctggt tccggtgtac
     4381 tcggggaagg ttaaaagcag tttgcgtttg atcccagatg atctatccct cctgaaactc
     4441 taccctccag gggatgagga agaggcagag ctggcagatt cgagtggtct ctaccatgag
     4501 ggctccccat caccaggctc tccctggaag acaaagctga ggactaagga taaagaagag
     4561 aagaaaaaga cagagtttct ggatctggac aactctcctc tgtccccacc ttcaccaagg
     4621 accaaaagca gaacgcatac tcgggcactc aagaagttaa gtgaggtgaa caagcgcctc
     4681 caggatctcc gttcctgtct gagccccaag ccacctcagg gtcaagagca acagggccaa
     4741 gaggatgaag tggtcttggt ggaagggccc accctcccag agaccccccg actcttccca
     4801 ctcaaaatcc gttgccgggc tgacctggtc agattgcccc tcaggatgtc ggagcccctg
     4861 cagagtgtgg tggaccacat ggccacccac cttggggtgt ccccaagcag gatccttttg
     4921 ctttttggag agacagagct atcacctact gccactccca ggaccctaaa gctcggagtg
     4981 gctgacatca ttgactgtgt ggtactaaca agttctccag aggccacaga gacgtcccaa
     5041 cagctccagc tccgggtgca gggaaaggag aaacaccaga cactggaagt ctcactgtct
     5101 cgagattccc ctctaaagac cctcatgtcc cactatgagg aggccatggg actgtcggga
     5161 cggaagctct ccttcttctt tcgtgggaca aagctttcag gcagggagct gccagctgac
     5221 ctgggcatgg aatctgggga cctcattgag gtctggggct gagcggccgc tcgagtctag
     5281 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5341 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5401 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     5461 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     5521 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     5581 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5641 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5701 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5761 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta
     5821 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa
     5881 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     5941 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     6001 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     6061 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     6121 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     6181 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     6241 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     6301 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     6361 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     6421 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     6481 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     6541 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     6601 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     6661 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     6721 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     6781 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     6841 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca
     6901 cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     6961 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     7021 ccaccccaac ttgtttattg cagcttataa tg
//
