LOCUS       sequence-362292-        7682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for knock-in of p2a-CreERT2 into the medaka col2a1a 
            locus via CRISPR/Cas9-mediated homology directed repair.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7682)
  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 7682)
  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-col2a1a-KI-donor
FEATURES             Location/Qualifiers
     source          1..7682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        305..323
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             687..743
                     /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             744..1772
                     /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             1785..2720
                     /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             1833..2726
                     /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    2806..2887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3098..3117
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(3189..3902,3903..3905,3906..3908))
                     /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(3192..3902,3903..3905,3906..3908))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3234..3255)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3583..3602
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(join(5514..5520,5521..5538,5539..5544))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    5559..5580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5700..5717)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5871..6459)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5951..5970)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6630..7421,7422..7490))
                     /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     7253..7272
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7491..7595)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7663..7681
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc
       61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc
      121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact
      181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt
      241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc
      301 ggcgtaatac gactcactat agggagagcg gccgccagat cttccggatg gctcgagttt
      361 ttcagcaaga tttacgccag tcgaccacct ctggaaccgc agcagcctct gctgtttcac
      421 cccatagccc ggccacagga aacctgaaga aggcgttggt tctgcaggga tccaatgatg
      481 tggaaatccg tgcagagggt aacagccgct tcacctacag tgtgatggag gatggctgca
      541 agaggcattc aggccggtgg agcaagaccg tctttgagta caaaacaatg aaaacctcac
      601 gcttgcccat agtcgacatt gctcccatgg acattggagg agcagatcag gaatttggag
      661 ttgacatagg ccccgtctgc tttttggcta caaacttcag cctgctgaag caggctggag
      721 acgtggagga gaaccctgga cctatgtcca atttactaac cgtacaccaa aatttgcctg
      781 cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg
      841 atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg
      901 cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg
      961 attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg
     1021 gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg
     1081 tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac
     1141 aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg
     1201 atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac
     1261 gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa
     1321 tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac
     1381 ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg
     1441 atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca
     1501 ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt
     1561 acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg
     1621 tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg
     1681 gtggctggac caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag
     1741 gggcaatggt gcgcctgctg gaagatggcg atctcgagcc atctgctgga gacatgagag
     1801 ctgccaacct ttggccaagc ccgctcatga tcaaacgctc taagaagaac agcctggcct
     1861 tgtccctgac ggccgaccag atggtcagtg ccttgttgga tgctgagccc cccatactct
     1921 attccgagta tgatcctacc agacccttca gtgaagcttc gatgatgggc ttactgacca
     1981 acctggcaga cagggagctg gttcacatga tcaactgggc gaagagggtg ccaggctttg
     2041 tggatttgac cctccatgat caggtccacc ttctagaatg tgcctggcta gagatcctga
     2101 tgattggtct cgtctggcgc tccatggagc acccagtgaa gctactgttt gctcctaact
     2161 tgctcttgga caggaaccag ggaaaatgtg tagagggcat ggtggagatc ttcgacatgc
     2221 tgctggctac atcatctcgg ttccgcatga tgaatctgca gggagaggag tttgtgtgcc
     2281 tcaaatctat tattttgctt aattctggag tgtacacatt tctgtccagc accctgaagt
     2341 ctctggaaga gaaggaccat atccaccgag tcctggacaa gatcacagac actttgatcc
     2401 acctgatggc caaggcaggc ctgaccctgc agcagcagca ccagcggctg gcccagctcc
     2461 tcctcatcct ctcccacatc aggcacatga gtaacaaagg catggagcat ctgtacagca
     2521 tgaagtgcaa gaacgtggtg cccctctatg acctgctgct ggaggcggcg gacgcccacc
     2581 gcctacatgc gcccactagc cgtggagggg catccgtgga ggagacggac caaagccact
     2641 tggccactgc gggctctact tcatcgcatt ccttgcaaaa gtattacatc acgggggagg
     2701 cagagggttt ccctgccaca gcttgatagg ttgatcacgc ggcaggttcc aaaagccaaa
     2761 gctagcaagg gcgaattcca gcacactggc ggccgttact agaatgcagt gaaaaaaatg
     2821 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     2881 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     2941 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga
     3001 tcagatctct tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     3061 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     3121 gtatcttatc atgtctggat ctacgtaata cgactcacta tagttctaga ggctcgagag
     3181 gggccgcttt acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     3241 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc ggactgggtg
     3301 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     3361 tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt gaagttcacc
     3421 ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt gtagttgtac
     3481 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     3541 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     3601 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     3661 tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact gcacgccgta ggtcagggtg
     3721 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     3781 agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt
     3841 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     3901 ctcaccatgg tcactgtctg ctttgctgtt ggtctgggct cctgggtcac tggcttacta
     3961 atggagtctt tatgtatgag gactcttatc aattgttctt ctataaaggt ctgcagtgtt
     4021 tctgttcgtc ccctacatgg acacccagag cctcctaaat acaggagccc tgataactgc
     4081 acaagtgctc agattccagc agggtggaaa atgagataaa gtgtgcagat ggggaggggg
     4141 acgtgaatga gagatttgag ggatgaaagg gatggatgaa cgcattgaaa atagcccctt
     4201 tcacaaaata ataccagtaa attgtcatat aattaatgac cagaccttta cctgtaaatg
     4261 atgtgatgtt catatacaga agaatgccaa caatttacag atgattttac aaattcttaa
     4321 ttttcctttt ctaaatggat ttgttcacac attatctctt caaagcaatt gaattttctg
     4381 gaaaagattg gctgtgtgtg taaaaggtgc tactgaggaa tttaatgtga catggaatga
     4441 agcaaacagc agcctaagac aggatggcag gaaaaatgtc acatgtttaa aataagagtg
     4501 aaaccaacga cctggactta tagagactgt gtgtccttag gcaaagctct tgtacaaaca
     4561 gtgcttgtga ctgtgaaatt cggggtttgc ctggattgtg ttaaatgtgt gcttggatgt
     4621 atcataaaaa agaatcggtt ctctgtattt gacattttct gtaatttatc taaagttaca
     4681 actggctatg cctgattttt atcactattg aatcaatcat tttaaagaat gttaagtctg
     4741 ctgatttgga gagctcattt acatttattt tgaatgtctc ttatttaaca caactgattt
     4801 aagctttagt tgagtattgc aacggccccc caaaaacaca ggaaataaac aaactcaacc
     4861 aagaaaacat aacactcttc cacaataaaa gaagactgga aatctaaaaa aaaaaaagaa
     4921 aaaaaaagaa aattgacact tttattttct ctcacaacga agtgctctcc atgttggatg
     4981 ttggcactta agggcaccgg caatagagca cttttttgcc gttccggctg ctgcggttcc
     5041 agaggtggag tgagggtacc aagcttgatg catagcttga tctttctaga agatctccta
     5101 caatattctc agctgccatg gaaaatcgat gttcttcttt tattctctca agattttcag
     5161 gctgtatatt aaaacttata ttaagaacta tgctaaccac ctcatcagga accgttgtag
     5221 gtggcgtggg ttttcttggc aatcgactct catgaaaact acgagctaaa tattcaatat
     5281 gttcctcttg accaacttta ttctgcattt tttttgaacg aggtttagag caagcttcag
     5341 gaaactgaga caggaatttt attaaaaatt taaattttga agaaagttca gggttaatag
     5401 catccatttt ttgctttgca agttcctcag cattcttaac aaaagacgtc tcttttgaca
     5461 tgtttaaagt ttaaacctcc tgtgtgaaat tattatccgc tcataattcc acacattata
     5521 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5581 attgcgttgc gctcactgcc aattgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5641 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5701 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5761 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5821 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5881 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5941 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6001 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6061 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6121 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6181 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6241 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6301 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6361 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6421 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6481 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6541 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6601 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6661 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6721 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6781 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6841 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6901 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6961 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7021 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7081 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7141 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7201 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7261 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7321 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7381 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7441 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7501 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7561 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7621 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7681 cc
//
