LOCUS       sequence-182266-        6505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Murine Atoh1 with S325A mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6505)
  AUTHORS   Cheng YF, Tong M, Edge AS
  TITLE     Destabilization of Atoh1 by E3 Ubiquitin Ligase Huwe1 and Casein 
            Kinase 1 Is Essential for Normal Sensory Hair Cell Development.
  JOURNAL   J Biol Chem. 2016 Sep 30;291(40):21096-21109. Epub 2016 Aug 19.
  PUBMED    27542412
REFERENCE   2  (bases 1 to 6505)
  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: "J Biol 
            Chem."; date: "2016-09-30"; volume: "291(40)"; pages: "21096-21109. 
            Epub 2016 Aug 19"
COMMENT     Sequence Label: FLAG-Atoh1 S325A
FEATURES             Location/Qualifiers
     source          1..6505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             961..1008
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     primer_bind     complement(2096..2113)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2102..2326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2372..2800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2459..2478)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2669..2690
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2809..2829)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2814..3143
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2994..3129
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3056..3075
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3210..4004
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3264..3283)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3874..3893
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4178..4299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4215..4234)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4269..4288
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4348..4364)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4348..4364)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4361..4383)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4372..4388
                     /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(4396..4402,4403..4420,4421..4426))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4441..4462
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4579..4596)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4750..5338)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4830..4849)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5509..6300,6301..6369))
                     /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     6132..6151
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6370..6474)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcatg
      961 gactacaaag acgatgatga taaagattac aaagatgacg atgacaaggg gtcccgcctg
     1021 ctgcatgcag aagagtgggc tgaggtaaaa gagttggggg accaccatcg ccatccccag
     1081 ccgcaccacg tcccgccgct gacgccacag ccacctgcta ccctgcaggc gagagacctt
     1141 cccgtctacc cggcagaact gtccctcctg gatagcaccg acccacgcgc ctggctgact
     1201 cccactttgc agggcctctg cacggcacgc gccgcccagt atctgctgca ttctcccgag
     1261 ctgggtgcct ccgaggccgc ggcgccccgg gacgaggctg acagccaggg tgagctggta
     1321 aggagaagcg gctgtggcgg cctcagcaag agccccgggc ccgtcaaagt acgggaacag
     1381 ctgtgcaagc tgaagggtgg ggttgtagtg gacgagcttg gctgcagccg ccagcgagcc
     1441 ccttccagca aacaggtgaa tggggtacag aagcaaagga ggctggcagc aaacgcaagg
     1501 gaacggcgca ggatgcacgg gctgaaccac gccttcgacc agctgcgcaa cgttatcccg
     1561 tccttcaaca acgacaagaa gctgtccaaa tatgagaccc tacagatggc ccagatctac
     1621 atcaacgctc tgtcggagtt gctgcagact cccaatgtcg gagagcaacc gccgccgccc
     1681 acagcttcct gcaaaaatga ccaccatcac cttcgcaccg cctcctccta tgaaggaggt
     1741 gcgggcgcct ctgcggtagc tggggctcag ccagccccgg gagggggccc gagacctacc
     1801 ccgcccgggc cttgccggac tcgcttctca ggcccagctt cctctggggg ttactcggtg
     1861 cagctggacg ctttgcactt cccagccttc gaggacaggg ccctaacagc gatgatggca
     1921 cagaaggacc tgtcgccttc gctgcccggg ggcatcctgc agcctgtaca ggaggacaac
     1981 gccaaaacat ctcccagatc ccacagaagt gacggagagt tttcccccca ctctcattac
     2041 agtgactctg atgaggccag ttagtctaga gggcccgttt aaacccgctg atcagcctcg
     2101 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2161 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2221 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2281 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     2341 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2401 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2461 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2521 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2581 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2641 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2701 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2761 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     2821 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2881 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2941 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3001 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3061 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     3121 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     3181 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     3241 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     3301 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     3361 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     3421 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     3481 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     3541 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     3601 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     3661 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     3721 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     3781 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     3841 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3901 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3961 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     4021 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     4081 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4141 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4201 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4261 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     4321 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     4381 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     4441 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4501 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4561 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     4621 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     4681 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     4741 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     4801 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     4861 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4921 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4981 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     5041 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     5101 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     5161 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     5221 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     5281 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     5341 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     5401 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     5461 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     5521 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5581 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     5641 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     5701 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5761 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5821 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5881 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5941 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     6001 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     6061 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     6121 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     6181 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     6241 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     6301 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     6361 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     6421 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6481 tccccgaaaa gtgccacctg acgtc
//
