LOCUS       sequence-366330-        7938 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for knock-in of p2a-CreERT2 into the medaka osr1 
            locus via CRISPR/Cas9-mediated homology directed repair.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7938)
  AUTHORS   Hui TW, Winkler C
  TITLE     A novel non-disruptive and efficient knock-in allows fate tracing of
            resident osteoblast progenitors during repair of vertebral lesions 
            in medaka.
  JOURNAL   Development. 2022 May 20. pii: 275483. doi: 10.1242/dev.200238.
  PUBMED    35593425
REFERENCE   2  (bases 1 to 7938)
  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: 
            "Development. 2022 May 20. pii: 275483. doi: 10.1242/dev.200238."
COMMENT     Sequence Label: pJET-osr1-KI-donor
FEATURES             Location/Qualifiers
     source          1..7938
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(18..36)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        104..208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(209..277,278..1069)
                     /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(427..446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1240..1828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1729..1748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1982..1999
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2119..2140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2155..2160,2161..2178,2179..2185)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3165..3221
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3222..4250
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             4263..5198
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             4311..5204
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATA"
     polyA_signal    5284..5365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5576..5595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5667..6380,6381..6383,6384..6386))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5670..6380,6381..6383,6384..6386))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5712..5733)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6061..6080
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(7632..7650)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 attcggctgc aggggcggcc tcgtgatacg cctattttta taggttaatg tcatgataat
       61 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      121 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      181 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      241 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      301 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      361 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      421 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
      481 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
      541 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
      601 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
      661 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
      721 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
      781 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
      841 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
      901 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
      961 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1021 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1081 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1141 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1201 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1261 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1321 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1381 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1441 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     1501 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     1561 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     1621 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     1681 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     1741 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     1801 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     1861 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     1921 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     1981 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2041 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcaattgg
     2101 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     2161 ctttatgctt ccggctcgta taatgtgtgg aattatgagc ggataataat ttcacacagg
     2221 aggtttaaac tttaaacatg tcaaaagaga cgtcttttgt taagaatgct gaggaacttg
     2281 caaagcaaaa aatggatgct attaaccctg aactttcttc aaaatttaaa tttttaataa
     2341 aattcctgtc tcagtttcct gaagcttgct ctaaacctcg ttcaaaaaaa atgcagaata
     2401 aagttggtca agaggaacat attgaatatt tagctcgtag ttttcatgag agtcgattgc
     2461 caagaaaacc cacgccacct acaacggttc ctgatgaggt ggttagcata gttcttaata
     2521 taagttttaa tatacagcct gaaaatcttg agagaataaa agaagaacat cgattttcca
     2581 tggcagctga gaatattgta ggagatcttc tagaaagatt tacgccagtc gaccacctct
     2641 ggaaccgcag cagcctctgc tgtttcaccc catagcctca gccattttta gcggacggag
     2701 tggaaatcca agttgtattt taccatgaga tctaacataa tccatagttc atggaatcag
     2761 tgataccaga actgagatcc tgagatcagc tgctgttcat ccttgataca aaagtccaaa
     2821 aatggtcttc agtttgtttt ttctttgctc atgaagtggt cactctgact tccctttaac
     2881 taacatgtta tccatgaatg gacgttgagc agaacatgtg atctcgtgca ctggaagaat
     2941 ttgctgcgat cagcacaggt gggtcagcca cagtgaaggt gttgacttca cagcagctgt
     3001 gggatgccct aacctgctgc ctttctccct gcaggtacat ccattccaaa gagaagccct
     3061 tcaaatgtca agagtgcgga aaaggcttct gtcagtccag aacactggct gtccacaaaa
     3121 cattacacat gcaggtcaaa gagctgaagc cagccaaggt caaggctaca aacttcagcc
     3181 tgctgaagca ggctggagac gtggaggaga accctggacc tatgtccaat ttactaaccg
     3241 tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc
     3301 tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa atgcttctgt
     3361 ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg tttcccgcag
     3421 aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa
     3481 ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac
     3541 caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa aacgttgatg
     3601 ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt
     3661 cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg
     3721 cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac
     3781 gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg
     3841 caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg
     3901 tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg
     3961 ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag
     4021 caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt
     4081 ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac
     4141 cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta
     4201 acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat ctcgagccat
     4261 ctgctggaga catgagagct gccaaccttt ggccaagccc gctcatgatc aaacgctcta
     4321 agaagaacag cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg
     4381 ctgagccccc catactctat tccgagtatg atcctaccag acccttcagt gaagcttcga
     4441 tgatgggctt actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga
     4501 agagggtgcc aggctttgtg gatttgaccc tccatgatca ggtccacctt ctagaatgtg
     4561 cctggctaga gatcctgatg attggtctcg tctggcgctc catggagcac ccagtgaagc
     4621 tactgtttgc tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg
     4681 tggagatctt cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg
     4741 gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc
     4801 tgtccagcac cctgaagtct ctggaagaga aggaccatat ccaccgagtc ctggacaaga
     4861 tcacagacac tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc
     4921 agcggctggc ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggca
     4981 tggagcatct gtacagcatg aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg
     5041 aggcggcgga cgcccaccgc ctacatgcgc ccactagccg tggaggggca tccgtggagg
     5101 agacggacca aagccacttg gccactgcgg gctctacttc atcgcattcc ttgcaaaagt
     5161 attacatcac gggggaggca gagggtttcc ctgccacagc ttgataggtt gatcacgcgg
     5221 caggttccaa aagccaaagc tagcaagggc gaattccagc acactggcgg ccgttactag
     5281 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     5341 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     5401 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     5461 tgattatgat cctctagatc agatctcttg tttattgcag cttataatgg ttacaaataa
     5521 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     5581 ttgtccaaac tcatcaatgt atcttatcat gtctggatct acgtaatacg actcactata
     5641 gttctagagg ctcgagaggg gccgctttac ttgtacagct cgtccatgcc gagagtgatc
     5701 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
     5761 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     5821 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     5881 cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg
     5941 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     6001 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     6061 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc
     6121 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     6181 acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     6241 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     6301 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     6361 aacagctcct cgcccttgct caccatggtc actgtctgct ttgctgttgg tctgggctcc
     6421 tgggtcactg gcttactaat ggagtcttta tgtatgagga ctcttatcaa ttgttcttct
     6481 ataaaggtct gcagtgtttc tgttcgtccc ctacatggac acccagagcc tcctaaatac
     6541 aggagccctg ataactgcac aagtgctcag attccagcag ggtggaaaat gagataaagt
     6601 gtgcagatgg ggagggggac gtgaatgaga gatttgaggg atgaaaggga tggatgaacg
     6661 cattgaaaat agcccctttc acaaaataat accagtaaat tgtcatataa ttaatgacca
     6721 gacctttacc tgtaaatgat gtgatgttca tatacagaag aatgccaaca atttacagat
     6781 gattttacaa attcttaatt ttccttttct aaatggattt gttcacacat tatctcttca
     6841 aagcaattga attttctgga aaagattggc tgtgtgtgta aaaggtgcta ctgaggaatt
     6901 taatgtgaca tggaatgaag caaacagcag cctaagacag gatggcagga aaaatgtcac
     6961 atgtttaaaa taagagtgaa accaacgacc tggacttata gagactgtgt gtccttaggc
     7021 aaagctcttg tacaaacagt gcttgtgact gtgaaattcg gggtttgcct ggattgtgtt
     7081 aaatgtgtgc ttggatgtat cataaaaaag aatcggttct ctgtatttga cattttctgt
     7141 aatttatcta aagttacaac tggctatgcc tgatttttat cactattgaa tcaatcattt
     7201 taaagaatgt taagtctgct gatttggaga gctcatttac atttattttg aatgtctctt
     7261 atttaacaca actgatttaa gctttgtcaa gcgaccggga cactggaaaa acacgaccaa
     7321 agatgaccca aagaacagcc aaagactttt ccttctcggg aggcaaacat tcctcaagga
     7381 tgaaatattc caccggccac gtcttaaaga aaaaacagaa tcagcctcag tgcttgttat
     7441 caaagcctga cttggcagag aagccggtgg atgtgtggaa tgtgaaatgg accttgaaaa
     7501 acccctgtgg agcacttttt tgccgttccg gctgctgcgg ttccagaggt ggagtgaggg
     7561 taccaagctt gatgcatagc ttgatcttgc tgaaaaactc gagccatccg gaagatctgg
     7621 cggccgctct ccctatagtg agtcgtatta cgccggatgg atatggtgtt caggcacaag
     7681 tgttaaagca gttgatttta ttcactatga tgaaaaaaac aatgaatgga acctgctcca
     7741 agttaaaaat agagataata ccgaaaactc atcgagtagt aagattagag ataatacaac
     7801 aataaaaaaa tggtttagaa cttactcaca gcgtgatgct actaattggg acaattttcc
     7861 agatgaagta tcatctaaga atttaaatga agaagacttc agagcttttg ttaaaaatta
     7921 tttggcaaaa ataatata
//
