LOCUS       sequence-227654-        6145 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for precise CRISPR directed genomic integration using the 
            GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6145)
  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 6145)
  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-Stop-cmlc2-eGFP
FEATURES             Location/Qualifiers
     source          1..6145
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     224..241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(271..282,283..445)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    532..553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(568..573,574..591,592..598)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    606..622
                     /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     611..633
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     630..646
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..646
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             642..956
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSIMIGSND
                     "
     promoter        667..685
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(733..751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(761..778)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(761..777)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(770..792)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2725..2745
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2749..2751,2752..2754,2755..3468)
                     /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             join(2749..2751,2752..2754,2755..3465)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2794..2815)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3055..3074)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3402..3423
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3473..3697
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3767..3848
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3972..4053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4370..4388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4411..4598,4599..4610)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4664..4683)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4675..5484
                     /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(4742..4761)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(5627..6145,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6116..6135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
       61 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      121 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      181 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      241 ggaagcggaa cagatttaaa tggtaccgag cagaggtgta aaaagtactc aaaaatttta
      301 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
      361 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
      421 aaggaagtaa aagtaaaagc aagaaagaaa actagagtgg tctcccttta gtgagggtca
      481 attgatggga ggcgttcggg ccacagcgga cacgcaggtg cgcaacgcaa ttaatgtgag
      541 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      601 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      661 gcgcgcaatt aaccctcact aaagggaaca aaagctggag gacctcgagg gggggcccgg
      721 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
      781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      901 gcctgaatgg cgaatggacc tgccacactt ctataatgat aggtagtaat gattagtaat
      961 gagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg
     1021 ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg
     1081 cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg
     1141 tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca
     1201 gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag
     1261 tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat
     1321 ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg
     1381 agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa tgcaaaaaaa
     1441 tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt attccaacac
     1501 gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt tttgtcaccc
     1561 tcacaggcaa tttagtataa tatattatat atatatatat atcatataat aatattcagt
     1621 ataatatata tatatatatc atattataat attcagtata atataaaaca caaacacata
     1681 tatgtataat ataatataac atttttattt attgagatgc ctctatggac cgtgttataa
     1741 gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat gttaaattaa
     1801 gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg cctccatgac
     1861 caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac tttgctgaat
     1921 atagctggct tgacagttat ctatgactct ataaatatat atatattttt tttttataaa
     1981 atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc cctcactccc
     2041 gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa gttattaaca
     2101 tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag cttgagatcc
     2161 tagtcaccgg cggcattcat ccatcctttt catccctcaa atctctcatt cacgtccccc
     2221 tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca cttgtgcagt
     2281 tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga acagaaacac
     2341 tgcagacctt tatagaagaa caaatgataa gagtcctcat acataaagac tccattagaa
     2401 acgtcagtga cccaggagcc cagaccaaca gcaaagcaga cagtgaagga tccacagcct
     2461 gactaactcg acaaaccccc ccaaacctaa ggtgagttga tctttaagct ttttacattt
     2521 tcagctcgca tatatcaatt cgcgcgcatt gtcacactag cacccactag cggtcagact
     2581 gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt
     2641 gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc
     2701 ggaattcacc tcgagaccat ggctccaaag aagaagcgta aggtaaccat ggtgagcaag
     2761 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2821 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2881 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2941 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     3001 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3061 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3121 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3181 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     3241 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3301 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     3361 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3421 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag atctgtgcct
     3481 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3541 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3601 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3661 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccaaa
     3721 gggcgaatta attcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     3781 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     3841 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg
     3901 aggtttttta aagcaagtaa aacctctaca aatgtggtca ttgatgagtt tggacaaacc
     3961 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4021 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4081 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4141 ggtcattcag atcaattaac cctcacccgg gctagcgatg catgggagct ctgacgtacg
     4201 ttgtctagca aggaagtgaa gagctatccg gatatatatg tatgttatca taactgtact
     4261 ggctatgtca ggtggtaatg actgtaacgt tacgttactc gttgtaggca cgacattgaa
     4321 tatgctcttc tccgctgtgg cccgaacgcc tcccgctagc gatgggatct aatacgactc
     4381 actataggga gaccaaggga aaatagaatg aagtgatctc caaaaaataa gtactttttg
     4441 actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa tgtaattaag
     4501 taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc ccaaaaataa
     4561 tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg ggatccagct
     4621 gcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     4681 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     4741 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     4801 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     4861 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     4921 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     4981 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     5041 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     5101 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     5161 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     5221 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     5281 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     5341 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     5401 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     5461 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcaaaaa
     5521 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5581 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5641 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5701 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5761 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5821 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5881 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5941 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6001 gatacctaca gcgtgagcta tgagaaagcg ccacggttcc cgaagggaga aaggcggaca
     6061 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6121 acgcctggta tctttatagt cctgt
//
