LOCUS       sequence-250993-        5993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR (donor) Plasmid for Inserting PuroR-2A-GFP-AID into the 5' end 
            of the Mouse Npm1 Gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5993)
  TITLE     Plasmids for generating AID/Degron Cell Lines in Human and Mouse
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5993)
  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: 
            "Unpublished"
COMMENT     Sequence Label: Mouse 5' Npm1 AID GFP PuroR
FEATURES             Location/Qualifiers
     source          1..5993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             997..1596
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MATEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1496..1516
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1603..1656
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1666..1668,1669..2379)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1708..1729)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2316..2337
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2389..3069
                     /codon_start=1
                     /gene="Arabidopsis thaliana AXR3"
                     /product="auxin-responsive IAA17 protein"
                     /label=auxin-inducible degron
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="GSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNNE
                     PANKEGSTTHDVVTFDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGG
                     PEAAAFVKVSMDGAPYLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMNE
                     RKLMDLVNSWDYVPSYEDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAMEK
                     CKSRA"
     primer_bind     complement(4154..4170)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(4220..4808)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4300..4319)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4979..5770,5771..5839))
                     /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     5602..5621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5840..5944)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cattggtacc attatctaca ttcctccccc aaattcttaa atatgctagc tccatgttga
       61 gataaaaaga ttagggccgc tgtcagaaag cacccaacgc tgagggatgt tcgtcaggat
      121 tcattttaaa gcgctaattc ccagccgaag attcgtatta aggctcatga atttctaaaa
      181 aagcgatagg ccttcatttg gaatgatgaa gacggcctgc gttttctcag ttctgcacgc
      241 tctaaagttt tagaaagatg ctctttggag gcgactgact gggtttctct ttggtcccca
      301 aatgacataa agctgataat tttcgttttg aaaatgctgc ctcagaaaag aggtgcaaaa
      361 gcatggctgc ggggaaagta gcctagatcc agccactccg ccctgggctc ctgaagctac
      421 gtgaaaacga agagcaagcc ttcgagcaag ccaaatcctg aggtgattat gcttgttcca
      481 ggcctacggg gaacggaggg tggagaaaag agacgctctg tggttccctg gctgcagccg
      541 ccatctttca gggtgggctg cgcacgctct cggccctggg cccgcgagca cgagcggaga
      601 agacgtggaa gccctgaaaa cccagagcga ggccacaaag agaaaacgcg cctgcgcttg
      661 ggcgacagta gtggagggac ggggccagag acgaacgcgc gtgcgcagtg agagcccgcc
      721 aagcacctgg aggaggaggg gggcgaggta cctaggcgtg aggatgaaag gcgcacgcgc
      781 aaaagcgcga ctgtgcgggg agtgggaggg atccagagga ggctcgcgga ggcaggacta
      841 gcgcagatct cgcgagatct cctgcgacca tatatacgtg cggagagccc gcgtcctttc
      901 cttggcgtga ttccgtcctg cgcgtctgtt ctgtggaaca ggaggcagtt gttttccgtc
      961 cggcttctcc cacaccgaag tgcgcgcctc cacctcatgg ccaccgagta caagcccacg
     1021 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     1081 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     1141 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     1201 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     1261 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     1321 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     1381 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     1441 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     1501 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     1561 ccgcgcacct ggtgcatgac ccgcaagccc ggtgccagcg gtgaaggcag aggctctctc
     1621 ctcacatgtg gggatgttga agaaaatcca ggtcccatgt ctagcgtgag caagggcgag
     1681 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     1741 aagttcagcg tgcgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag
     1801 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     1861 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     1921 tccgccatgc ccgaaggcta cgtccaggag cgcaccatca ccttcaagga cgacggcacc
     1981 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2041 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacttc
     2101 aacagccaca acgtctatat taccgccgac aagcagaaga acggcatcaa ggccaacttc
     2161 aagatccgcc acaacgtgga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2221 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2281 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2341 gccgccggga tcactcacgg catggacgag ctgtacaagg gaggtaccgg cagcgtggaa
     2401 ctgaacctgc gcgagacaga gctgtgcctg ggactgcctg gcggcgatac agtggctcct
     2461 gtgaccggca acaagagagg cttcagcgag acagtggacc tgaagctgaa cctgaacaac
     2521 gagcccgcca acaaagaggg cagcaccacc cacgacgtcg tgaccttcga cagcaaagag
     2581 aagtccgcct gccccaagga ccccgccaag cctcctgcta aagctcaggt cgtgggatgg
     2641 cctcccgtgc ggagctacag aaagaacgtg atggtgtcct gccagaagtc ctctggcggc
     2701 cctgaggctg ccgccttcgt gaaagtgtct atggatggcg ccccttacct gagaaagatc
     2761 gacctgagaa tgtacaagag ctacgacgag ctgagcaacg ccctgagcaa catgttcagc
     2821 agcttcacaa tgggcaagca cggcggcgag gaaggcatga tcgacttcat gaacgagcgg
     2881 aagctgatgg acctcgtgaa cagctgggac tacgtgccca gctacgagga caaggacggc
     2941 gactggatgc tcgtgggaga tgtgccctgg cccatgttcg tggacacatg caagagactg
     3001 agactgatga agggcagcga cgccatcggc ctggccccca gagctatgga aaagtgcaag
     3061 agcagagccg gagggtctgg aggctctgga tccgaagact cgatggatat ggacatgagt
     3121 cctcttaggc ctcagaacta ccttttcggt aactgactgg agggggaagc gtgtgcaggg
     3181 cctggtgatg gggggggggg cgggagccca gccgaggcct ggcgtgtggg gaacgcggcg
     3241 taggtggagg ccggagagca aggaggaatc tcccaggcct tagaagcagc ggcctgaaca
     3301 ctaggaaagc cgccggaaaa tggggcggga gcggcccagc ctgtagcctc gggtgttagg
     3361 agcggccggc cggccggcga gggtgcgacc ttgcctgtgg cgcgaacttg agaggcgttt
     3421 ctcctgtgca cgaaagcccg cacggtcgct tccgagctcc tttttttcct cttgtgttga
     3481 gccacggcag gcggcacggg agaaaggggc ctgtggagcg aggcgaggcc tacctccgcc
     3541 ctgttgggga ggatacatct gcaggcgctg ggctttccag gggccgcatc ttgtattctg
     3601 tggcgaccga gggcttcgcc cagtaacggg gaatggtccg agcaggcgcc ctgaggcaga
     3661 aggggcagca acccctagga gcacgtggtt gccacgtggt tgggggttag ggagggggtg
     3721 tgggctctac atccgggctc tccgactcta gccaaccttc agccgtggga tcttgtggct
     3781 tagttaaaag ttctgggagg gaaatgttta tttcatagcc cgtggcgtat gggtttttga
     3841 gatctttgtt catatctttc agtatccaca gtgatggagc cccccctttt taatttgcct
     3901 ctggtaggcg gagtcagcat cacgttgcag atagctgtac tgatggccca cgtggcttaa
     3961 tgttaggagt acccttaaat catctacctg gctctatcta gaactacttt ttcttggggt
     4021 ggggagaggg aaggatctgt tgaacagaca agattcgctt tagcatcaag gcttttgggg
     4081 atcacatcta tagggcgcgc ccctcgagct agaagatctc ctagaggtac cctagagaat
     4141 tcctagagct aacactggcc gtcgttttac cggtccgtgc aaaaggccag aaccgtaaaa
     4201 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4261 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4321 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4381 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4441 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4501 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4561 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4621 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4681 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4741 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4801 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4861 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4921 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4981 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5041 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5101 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5161 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5221 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5281 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5341 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5401 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5461 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5521 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5581 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5641 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5701 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5761 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5821 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5881 attgtctcgt gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5941 cgcgcacatt tccccgaaaa gtgccacctg acgtcaaaca gctatgacca tgg
//
