LOCUS       sequence-220615-        7858 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  targeting osTIR1-GFP into mouse Actb locus for auxin inducible 
            degradation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7858)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 7858)
  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 
            Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: PBS Actb-2A-TIR1-GFP
FEATURES             Location/Qualifiers
     source          1..7858
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     complement(1160..1179)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1161..1179)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1189..1206)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1189..1205)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1198..1220)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(1346..1801)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1483..1504)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1695..1714
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1828..1932
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1933..2001,2002..2793)
                     /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(2151..2170)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2964..3552
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3453..3472
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3706..3723
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3840..3861
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3876..3881,3882..3899,3900..3906)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3914..3930
                     /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     3919..3941
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3938..3954
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3938..3954
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3973..3993
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3975..3993
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4022..4038
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4023..4039
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5118..5171
                     /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             5178..6902
                     /codon_start=1
                     /product="rice transport inhibitor response 1 protein"
                     /label=OsTIR1
                     /note="F-box protein that mediates auxin-dependent 
                     interaction of a ubiquitin ligase with an auxin-inducible 
                     degron (Tan et al., 2007)"
                     /translation="MTYFPEEVVEHIFSFLPAQRDRNTVSLVCKVWYEIERLSRRGVFV
                     GNCYAVRAGRVAARFPNVRALTVKGKPHFADFNLVPPDWGGYAGPWIEAAARGCHGLEE
                     LRMKRMVVSDESLELLARSFPRFRALVLISCEGFSTDGLAAVASHCKLLRELDLQENEV
                     EDRGPRWLSCFPDSCTSLVSLNFACIKGEVNAGSLERLVSRSPNLRSLRLNRSVSVDTL
                     AKILLRTPNLEDLGTGNLTDDFQTESYFKLTSALEKCKMLRSLSGFWDASPVCLSFIYP
                     LCAQLTGLNLSYAPTLDASDLTKMISRCVKLQRLWVLDCISDKGLQVVASSCKDLQELR
                     VFPSDFYVAGYSAVTEEGLVAVSLGCPKLNSLLYFCHQMTNAALVTVAKNCPNFTRFRL
                     CILEPGKPDVVTSQPLDEGFGAIVRECKGLQRLSISGLLTDKVFMYIGKYAKQLEMLSI
                     AFAGDSDKGMMHVMNGCKNLRKLEIRDSPFGDAALLGNFARYETMRSLWMSSCNVTLKG
                     CQVLASKMPMLNVEVINERDGSNEMEENHGDLPKVEKLYVYRTTAGARDDAPNFVKIL"
     CDS             6930..6959
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6966..6995
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(7023..7025,7026..7739)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7023..7025,7026..7736)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(7065..7086)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7326..7345)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7673..7694
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tttttgtttt gttttggcgc ttttgactca ggatttaaaa actggaacgg tgaaggcgac
       61 agcagttggt tggagcaaac atcccccaaa gttctacaaa tgtggctgag gactttgtac
      121 attgttttgt tttttttttt ttggttttgt ctttttttaa tagtcattcc aagtatccat
      181 gaaataagtg gttacaggaa gtccctcacc ctcccaaaag ccacccccac tcctaagagg
      241 aggatggtcg cgtccatgcc ctgagtccac cccggggaag gtgacagcat tgcttctgtg
      301 taaattatgt actgcaaaaa tttttttaaa tcttccgcct taatacttca tttttgtttt
      361 taatttctga atggcccagg tctgaggcct cccttttttt tgtcccccca acttgatgta
      421 tgaaggcttt ggtctccctg ggagggggtt gaggtgttga ggcagccagg gctggcctgt
      481 acactgactt gagaccaata aaagtgcaca ccttacctta cacaaacagc ttgtggctct
      541 gtggctttgc tgggtgtggg gagcaggttg ggtgggtgtg gagctctatt ggggggggca
      601 tctagggtgg gctaggcctt gctgatggta tctagtggga gggctacagg ccctttaatg
      661 aagtctcaca aataaccaca ccttcagact gtcttgaaga gttgaaaagg tactcctcca
      721 gaaacctagg ccaagttggc ctgcactaac actaccttcc tcaaccgagg ggtgtccaca
      781 ccctttgcct ccatctgcat aactgggttt caagcccctc tgctagtctg aagctgccct
      841 ttccagagct gtgactgaca gcaggaaggt gtgaagccta tggcttcctt cccagggatg
      901 ggcatctttt ccctggtttt gttttgagag cctaggactg aggaacacct cagagggagg
      961 aagggggtca tcctggaaat cagccccttc cacatggcac gcagctccag cctttgtcag
     1021 tcgtgatcct tggacttggg tgccgcaagt tagctttgac tgggatcatt tgttcacaca
     1081 tgcagagaac actggttggt tgagtcctag tcatctccat ccagctatgc ggccgccacc
     1141 gcggtggagc tccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     1201 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     1261 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     1321 gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atattttgtt aaaattcgcg
     1381 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     1441 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     1501 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     1561 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     1621 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     1681 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     1741 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     1801 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1861 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     1921 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     1981 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     2041 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     2101 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     2161 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     2221 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     2281 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     2341 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     2401 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     2461 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     2521 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     2581 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     2641 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     2701 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     2761 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     2821 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     2881 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2941 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     3001 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     3061 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     3121 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     3181 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     3241 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     3301 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     3361 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     3421 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3481 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3541 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3601 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3661 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3721 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     3781 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     3841 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     3901 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     3961 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctgggtac cgggcccccc
     4021 ctcgaggtcg acggtatcga taagcttcag tttcacctgc cctgagtgtt tcttgtggct
     4081 ttctgaactt gacaacatta tttatttttc tctacagatc atgtttgaga ccttcaacac
     4141 cccagccatg tacgtagcca tccaggctgt gctgtccctg tatgcctctg gtcgtaccac
     4201 aggcattgtg atggactccg gagacggggt cacccacact gtgcccatct acgagggcta
     4261 tgctctccct cacgccatcc tgcgtctgga cctggctggc cgggacctga cagactacct
     4321 catgaagatc ctgaccgagc gtggctacag cttcaccacc acagctgaga gggaaatcgt
     4381 gcgtgacatc aaagagaagc tgtgctatgt tgctctagac ttcgagcagg agatggccac
     4441 tgccgcatcc tcttcctccc tggagaagag ctatgagctg cctgacggcc aggtcatcac
     4501 tattggcaac gagcggttcc gatgccctga ggctcttttc cagccttcct tcttgggtaa
     4561 gttgtagcct agtcctttct ccatctaaag gtgacaaaac tcctgaggcc atagtacaag
     4621 ttaagtctga tttctgtcac tcttctctta ggtatggaat cctgtggcat ccatgaaact
     4681 acattcaatt ccatcatgaa gtgtgacgtt gacatccgta aagacctcta tgccaacaca
     4741 gtgctgtctg gtggtaccac catgtaccca ggcattgctg acaggatgca gaaggagatt
     4801 actgctctgg ctcctagcac catgaagatc aaggtaagct aagcatcctt agcttggtga
     4861 gggtgggccc tgtggttgtc agagcaacct tctaggttta aggggaatcc cagcacccag
     4921 agagctcacc attcaccatc ttgtcttgct ttcttcagat cattgctcct cctgagcgca
     4981 agtactctgt gtggatcggt ggctccatcc tggcctcact gtccaccttc cagcagatgt
     5041 ggatcagcaa gcaggagtac gatgagtccg gcccctccat cgtgcaccgc aagtgcttca
     5101 agcttgatat cgaattcgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     5161 atcccggccc taccggtatg acgtacttcc cggaggaggt ggtggagcac atcttcagct
     5221 tcctgccggc gcagcgcgac cgcaacacgg tctcgctcgt ctgcaaggtg tggtacgaga
     5281 tcgagaggct gagccgccgc ggcgtcttcg tgggcaactg ctacgccgtg cgcgccggcc
     5341 gcgtcgccgc gcggttcccc aacgtgcggg cgctcacggt gaaggggaag ccccacttcg
     5401 ccgacttcaa cctcgtgccc cccgactggg gcggctacgc ggggccgtgg atcgaggcgg
     5461 ccgcgagggg atgccacggc ctggaggagc tcaggatgaa gcggatggtg gtgtccgacg
     5521 agagcctcga gctgctggct cgctcgttcc cgcggttcag ggctcttgtt cttatcagct
     5581 gcgaggggtt cagcactgac gggctagccg ccgtcgcgag ccattgcaag cttctgaggg
     5641 agttggattt gcaggaaaat gaagtggagg atcgagggcc taggtggctt tcctgcttcc
     5701 ctgattcctg cacatcactt gtctcattga attttgcctg catcaaaggg gaggttaatg
     5761 ctggttcact ggagagactt gttagcaggt ccccaaacct gcggagtttg aggctgaatc
     5821 gatctgtatc ggtagataca cttgcaaaga tactactgcg tacccctaac ttggaggatt
     5881 tggggacagg gaatttgaca gatgacttcc aaactgagtc ctactttaag cttaccagtg
     5941 ctctggagaa atgcaagatg ttgaggagtt tgtctggatt ctgggatgct tctcctgttt
     6001 gcctgtcatt tatctacccc ctgtgtgctc aactgacagg attgaacttg agctatgcac
     6061 ccacacttga tgcttctgac cttacaaaaa tgattagccg ctgtgtgaag ctccaacgcc
     6121 tttgggtact ggattgtatc tcggacaaag gcttgcaagt ggtggcctcc agttgcaaag
     6181 acttgcaaga actcagggta tttccatcag atttctacgt agctggttat tctgcagtga
     6241 cagaggaggg acttgttgca gtatccttgg gctgtccaaa actgaactca ctactgtact
     6301 tctgtcacca aatgactaat gctgcactag ttactgtcgc caagaactgt ccaaatttca
     6361 cacgattcag actttgtatt cttgagccag ggaagcctga tgttgtgaca agccaaccat
     6421 tagatgaagg ctttggagct attgttcgtg agtgcaaggg attacaacgt ttgtcaatat
     6481 ctggtcttct cacagacaaa gttttcatgt atattgggaa atatgcaaaa caacttgaga
     6541 tgctttctat agcatttgct ggtgacagtg ataagggtat gatgcatgtt atgaatggat
     6601 gcaagaattt aaggaaactg gagataagag atagcccgtt tggtgatgct gcactcttgg
     6661 ggaattttgc taggtacgag acaatgcgat ccctttggat gtcatcttgc aatgtcacgt
     6721 taaaggggtg ccaagtcctt gcgtcaaaga tgccgatgct caatgttgag gtcataaatg
     6781 agcgggatgg tagcaatgaa atggaggaaa accatggaga tctgcctaaa gtggagaaat
     6841 tatatgtgta ccgcacaact gctggggcga gggatgatgc accaaatttt gttaaaatcc
     6901 tagtcgactc cggttctgct gctagtggtg aacaaaagtt gatttctgaa gaagatttga
     6961 acggtgaaca aaagctaatc tccgaggaag acttgaacgg tgaaggtagc ggcagcggta
     7021 gcgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     7081 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     7141 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     7201 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     7261 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     7321 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     7381 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     7441 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     7501 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     7561 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     7621 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     7681 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtgag
     7741 cggactgtta ctgagctgcg ttttacaccc tttctttgac aaaacctaac ttgcgcagaa
     7801 aaaaaaaaaa taagagacaa cattggcatg gctttgtttt tttaaatttt ttttaaag
//
