LOCUS       sequence-229892-        6893 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 6893)
  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 6893)
  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-eGFP
FEATURES             Location/Qualifiers
     source          1..6893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(14..25,26..188)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    275..296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(311..316,317..334,335..341)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    349..365
                     /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     354..376
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     373..389
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     373..389
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             385..750
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSSRGV"
     promoter        410..428
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(476..494)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(504..521)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(504..520)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(513..535)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             680..736
                     /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             743..1453
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             3216..3236
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3240..3242,3243..3245,3246..3959)
                     /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(3240..3242,3243..3245,3246..3956)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3285..3306)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3546..3565)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3893..3914
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3964..4188
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4258..4339
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4463..4544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4861..4879
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4902..5089,5090..5101)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5155..5174)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5166..5975
                     /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(5233..5252)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6118..6706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6607..6626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6860..6877
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaatggtacc gagcagaggt gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa
       61 gtacttaggg aaaattttac tcaattaaaa gtaaaagtat ctggctagaa tcttacttga
      121 gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt attaaggaag taaaagtaaa
      181 agcaagaaag aaaactagag tggtctccct ttagtgaggg tcaattgatg ggaggcgttc
      241 gggccacagc ggacacgcag gtgcgcaacg caattaatgt gagttagctc actcattagg
      301 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      361 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      421 actaaaggga acaaaagctg gaggacctcg agggggggcc cggtacccaa ttcgccctat
      481 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
      541 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
      601 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
      661 acctgccaca cttcccggag ccacgaactt ctctctgtta aagcaagcag gagacgtgga
      721 agaaaacccc ggtcctctcg aggtgtctaa gggcgaggaa ctgattaagg agaacatgca
      781 catgaagctg tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg
      841 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
      901 tctccccttc gccttcgaca tcctggctac cagcttcatg tacggcagca gaaccttcat
      961 caaccacacc cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg
     1021 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     1081 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg
     1141 ccctgtgatg cagaagaaaa cactcggctg ggaggccaac accgagatgc tgtaccccgc
     1201 tgacggcggc ctggaaggca gaagcgacat ggccctgaag ctcgtgggcg ggggccacct
     1261 gatctgcaac ttcaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc
     1321 cggcgtctac tatgtggacc acagactgga aagaatcaag gaggccgaca aagagaccta
     1381 cgtcgagcag cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca
     1441 caaacttaat tgaaactagt aattaagtct cagccaccgt taactgaaca tgtcaaaacc
     1501 tgtggagact gttgagattt gatgttctga aaagataaag cctataaata aaatgttgcc
     1561 caaatttcct gcctgatgtt tttctttgtc tttgctacat ggctttgctg ctcggatcgg
     1621 ctcactctgt gtatgccacg ttcactttgt actctccttc tcacggtagg tttattattt
     1681 ttagatgtgc agttagtttc tgtgaaataa cacaccacac actgatattg tctgtgcatt
     1741 gacttggtga gtgcacattg tttttgatct tgacatattt atatttgatt gatcaggtga
     1801 actgtgtgaa tctaaagtgc tccatacaga tgttctgcat tgaaaatatt ctcattttat
     1861 tagtggaagt gagtgtatgc cacatccaat caatttcagc aaacacccca gtatgattta
     1921 atgcaaaaaa atgaaggtat caaacacgca ttactacttt gcagttaaat atttaacatt
     1981 tattccaaca cgaaaaaaag cagtaaataa cactttgaca aacacgtcag gacatcttat
     2041 ttttgtcacc ctcacaggca atttagtata atatattata tatatatata tatcatataa
     2101 taatattcag tataatatat atatatatat catattataa tattcagtat aatataaaac
     2161 acaaacacat atatgtataa tataatataa catttttatt tattgagatg cctctatgga
     2221 ccgtgttata agaagtaaag atcaggagaa gtaaacatga agtgtaatta tgaatactga
     2281 tgttaaatta agctatgatg agttttcact gttaatttac catctcaatt aaatgttgat
     2341 gcctccatga ccaagttaag cagatgagac tgaggacaac tgtagaagac aagatgttca
     2401 ctttgctgaa tatagctggc ttgacagtta tctatgactc tataaatata tatatatttt
     2461 ttttttataa aatgatttat ttataactat atatccattt ctcagacagg tgcttcatat
     2521 ccctcactcc cgtagctgtc catgctggat ctgtccccgt tgtttttaaa aagctaaata
     2581 agttattaac atgactgcat ccagcgagcc aaacctgtct ggtgtacagc taccagagaa
     2641 gcttgagatc ctagtcaccg gcggcattca tccatccttt tcatccctca aatctctcat
     2701 tcacgtcccc ctccccatct gcacacttta tctcattttc caccctgctg gaatctgagc
     2761 acttgtgcag ttatcagggc tcctgtattt aggaggctct gggtgtccat gtaggggacg
     2821 aacagaaaca ctgcagacct ttatagaaga acaaatgata agagtcctca tacataaaga
     2881 ctccattaga aacgtcagtg acccaggagc ccagaccaac agcaaagcag acagtgaagg
     2941 atccacagcc tgactaactc gacaaacccc cccaaaccta aggtgagttg atctttaagc
     3001 tttttacatt ttcagctcgc atatatcaat tcgcgcgcat tgtcacacta gcacccacta
     3061 gcggtcagac tgcagattgc agcacgaaac aggaagctga ctccacatgg tcacatgctc
     3121 actgaagtgt tgacttccct gacagctgtg cactttctaa accggttttc tcattcattt
     3181 acagttcagc cggaattcac ctcgagacca tggctccaaa gaagaagcgt aaggtaacca
     3241 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     3301 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     3361 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     3421 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     3481 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     3541 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     3601 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     3661 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     3721 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     3781 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     3841 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     3901 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     3961 gatctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4021 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4081 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4141 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     4201 aaagaaccaa agggcgaatt aattcattga tgagtttgga caaaccacaa ctagaatgca
     4261 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     4321 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     4381 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggtc attgatgagt
     4441 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     4501 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     4561 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     4621 tctacaaatg tggtcattca gatcaattaa ccctcacccg ggctagcgat gcatgggagc
     4681 tctgacgtac gttgtctagc aaggaagtga agagctatcc ggatatatat gtatgttatc
     4741 ataactgtac tggctatgtc aggtggtaat gactgtaacg ttacgttact cgttgtaggc
     4801 acgacattga atatgctctt ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc
     4861 taatacgact cactataggg agaccaaggg aaaatagaat gaagtgatct ccaaaaaata
     4921 agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa
     4981 atgtaattaa gtaaaagtaa aagtattgat ttttaattgt actcaagtaa agtaaaaatc
     5041 cccaaaaata atacttaagt acagtaatca agtaaaatta ctcaagtact ttacacctct
     5101 gggatccagc tgcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     5161 gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg
     5221 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     5281 atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     5341 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     5401 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     5461 tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     5521 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     5581 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     5641 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     5701 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     5761 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     5821 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     5881 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     5941 tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac
     6001 actggcaaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6061 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6121 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6181 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6241 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6301 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6361 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6421 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6481 caccgaactg agatacctac agcgtgagct atgagaaagc gccacggttc ccgaagggag
     6541 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6601 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6661 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6721 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6781 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     6841 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaacaga ttt
//
