LOCUS       sequence-416600-        6842 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible expression of N-terminally EGFP-tagged NIP45 delta-SLD2. 
            Use with Flp-In T-Rex system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6842)
  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 6842)
  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: EGFP-NIP45 delta-SLD2
FEATURES             Location/Qualifiers
     source          1..6842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(51..70)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        242..621
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        622..825
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     776..796
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    827..845
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    848..866
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     876..900
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(996..998,999..1001,1002..1712)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(996..998,999..1001,1002..1712)
                     /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(1041..1062)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1302..1321)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1649..1670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2801..2818)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2807..3031
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3164..3183)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3315..3362
                     /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             3370..4392
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3370..4392
                     /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    4522..4643
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4559..4578)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4613..4632
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4692..4708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4692..4708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4705..4727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4716..4732
                     /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(4740..4746,4747..4764,4765..4770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4785..4806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4923..4940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5094..5682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5174..5193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5853..6644,6645..6713))
                     /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     6476..6495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6714..6818)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
       61 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
      121 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
      181 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
      241 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      301 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      361 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      421 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      481 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      541 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      601 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      661 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      721 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      781 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat
      841 agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc
      901 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
      961 ctccggactc tagcgtttaa acttaagctt ccaccatggt gagcaagggc gaggagctgt
     1021 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1081 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1141 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1201 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1261 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1321 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1381 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1441 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1501 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1561 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga
     1621 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     1681 ggatcactct cggcatggac gagctgtaca agaagcttgg taccatggcg gagcctgtgg
     1741 ggaagcgggg ccgctggtcc ggaggtagcg gtgccggccg agggggtcgg ggcggctggg
     1801 gcggtcgggg ccggcgtcct cgggcccagc ggtctccatc ccggggcacg ctggacgtag
     1861 tgtctgtgga cttggtcacc gacagcgatg aggaaattct ggaggtcgcc accgctcgcg
     1921 gtgccgcgga cgaggttgag gtggagcccc cggagccccc ggggccggtc gcgtcccggg
     1981 ataacagcaa cagtgacagc gaaggggagg acaggcggcc cgcaggaccc ccgcgggagc
     2041 cggtcaggcg gcggcggcgg ctggtgctgg atccggggga ggcgccgctg gttccggtgt
     2101 actcggggaa ggttaaaagc agccttcgcc ttatcccaga tgatctatcc ctcctgaaac
     2161 tctaccctcc aggggatgag gaagaggcag agctggcaga ttcgagtggt ctctaccatg
     2221 agggctcccc atcaccaggc tctccctgga agacaaagct gaggactaag gataaagaag
     2281 agaagaaaaa gacagagttt ctggatctgg acaactctcc tctgtcccca ccttcaccaa
     2341 ggaccaaaag cagaacgcat actcgggcac tcaagaagtt aagtgaggtg aacaagcgcc
     2401 tccaggatct ccgttcctgt ctgagcccca agccacctca gggtcaagag caacagggcc
     2461 aagaggatga agtggtcttg gtggaagggc ccaccctccc agagaccccc cgactcttcc
     2521 cactcaaaat ccgttgccgg gctgacctgg tcagattgcc cctcaggatg tcggagcccc
     2581 tgcagagtgt ggtggaccac atggccaccc accttggggt gtccccaagc aggatccttt
     2641 tgctttttgg agagacagag ctatcaccta ctgccactcc caggacccta aagctcggag
     2701 tggctgacat cattgactgt gtggtactaa caagttctcc agaggccaca gagtgagcgg
     2761 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2821 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2881 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2941 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3001 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3061 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3121 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3181 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3241 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3301 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     3361 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     3421 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     3481 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     3541 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     3601 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     3661 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     3721 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     3781 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     3841 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     3901 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     3961 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     4021 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     4081 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     4141 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     4201 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     4261 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     4321 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     4381 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4441 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4501 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4561 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4621 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4681 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4741 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4801 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4861 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4921 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4981 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5041 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5101 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5161 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5221 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5281 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5341 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5401 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5461 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5521 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5581 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5641 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5701 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5761 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5821 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5881 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5941 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6001 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6061 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6121 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6181 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6241 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6301 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6361 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6421 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6481 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6541 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6601 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6661 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6721 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6781 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6841 cc
//
