LOCUS       sequence-222257-        2856 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2856)
  AUTHORS   Wierson WA, Welker JM, Almeida MP, Mann CM, Webster DA, Torrie ME, 
            Weiss TJ, Kambakam S, Vollbrecht MK, Lan M, McKeighan KC, Levey J, 
            Ming Z, Wehmeier A, Mikelson CS, Haltom JA, Kwan KM, Chien CB, 
            Balciunas D, Ekker SC, Clark KJ, Webber BR, Moriarity BS, Solin SL, 
            Carlson DF, Dobbs DL, McGrail M, Essner J
  TITLE     Efficient targeted integration directed by short homology in 
            zebrafish and mammalian cells.
  JOURNAL   Elife. 2020 May 15;9. pii: 53968. doi: 10.7554/eLife.53968.
  PUBMED    32412410
REFERENCE   2  (bases 1 to 2856)
  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: "Elife."; 
            date: "2020-05-15"; volume: "9. pii: 53968. doi"; pages: " 
            10.7554/eLife.53968"
COMMENT     Sequence Label: pPRISM-TagRFP-cmlc2-TagBFP
FEATURES             Location/Qualifiers
     source          1..2856
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        154..172
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(195..382,383..394)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(448..467)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             459..1268
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(526..545)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1411..1999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1900..1919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2153..2170
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(2200..2211,2212..2374)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    2461..2482
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2497..2502,2503..2520,2521..2527)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2535..2551
                     /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     2540..2562
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2559..2575
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2559..2575
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2571..2855
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCH"
     promoter        2596..2614
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(2662..2680)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2690..2707)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2690..2706)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2699..2721)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgaagagcta tccggatata tatgtatgtt atcataactg tactggctat gtcaggtggt
       61 aatgactgta acgttacgtt actcgttgta ggcacgacat tgaatatgct cttctccgct
      121 gtggcccgaa cgcctcccgc tagcgatggg atctaatacg actcactata gggagaccaa
      181 gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg
      241 taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt
      301 gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa
      361 tcaagtaaaa ttactcaagt actttacacc tctgggatcc agctgcatca tgaacaataa
      421 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
      481 cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
      541 gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc
      601 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
      661 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
      721 ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat
      781 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
      841 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
      901 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
      961 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac
     1021 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1081 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1141 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1201 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1261 ttttctaatc agaattggtt aattggttgt aacactggca aaaaggatct aggtgaagat
     1321 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1381 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1441 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1501 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1561 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1621 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1681 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1741 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1801 gctatgagaa agcgccacgg ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1861 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1921 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1981 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2041 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2101 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2161 agtgagcgag gaagcggaac agatttaaat ggtaccgagc agaggtgtaa aaagtactca
     2221 aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa ttaaaagtaa
     2281 aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt aaaattgtac
     2341 ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagtggt ctccctttag
     2401 tgagggtcaa ttgatgggag gcgttcgggc cacagcggac acgcaggtgc gcaacgcaat
     2461 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     2521 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     2581 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagg acctcgaggg
     2641 ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     2701 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2761 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2821 agttgcgcag cctgaatggc gaatggacct gccaca
//
