LOCUS       sequence-229888-        6967 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 6967)
  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 6967)
  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-gcry-mRFP
FEATURES             Location/Qualifiers
     source          1..6967
                     /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             3327..3347
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3351..4028
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3351..4028
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3481..3499)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3749..3768
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3943..3966
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4038..4262
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4332..4413
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4537..4618
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4935..4953
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4976..5163,5164..5175)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5229..5248)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5240..6049
                     /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(5307..5326)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6192..6780
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6681..6700
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6934..6951
                     /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 gcgttcctag ttctttaaac tgttacaaag tgtttggacc
     2701 agcaactgaa ttgcactgta aattaccaca tcagggtctc cggggtcaac gctgctaagc
     2761 aatggactgg aaaatttgtg acattgagtt tttggaataa ctgggaatgc tggataactc
     2821 tttgggtaaa gctatggaac aaaagtacca gcatgaacta agaaatgact gtcacagcac
     2881 aacatcaaac acaatgcaag acaaaggttc tctgcttttg ccttacttga cagaaaaagt
     2941 aagctctagg ttcatcgcat gcagacagca gcaatacatg acatatataa aggctggctc
     3001 ttttctccct tccattgaac tgaaacttcc actcagtcag acttgcgggg gatccacagc
     3061 ctgactaact cgacaaaccc ccccaaacct aaggtgagtt gatctttaag ctttttacat
     3121 tttcagctcg catatatcaa ttcgcgcgca ttgtcacact agcacccact agcggtcaga
     3181 ctgcagattg cagcacgaaa caggaagctg actccacatg gtcacatgct cactgaagtg
     3241 ttgacttccc tgacagctgt gcactttcta aaccggtttt ctcattcatt tacagttcag
     3301 ccggaattca cctcgagacc atggctccaa agaagaagcg taaggtaacc atggcctcct
     3361 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg
     3421 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     3481 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc
     3541 agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga
     3601 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg
     3661 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     3721 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg
     3781 aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga
     3841 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca
     3901 agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc
     3961 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg
     4021 gcgcctaaag ctgtgatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4081 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4141 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4201 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4261 ggcttctgag gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc
     4321 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4381 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     4441 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     4501 ggtcattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4561 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4621 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4681 aagcaagtaa aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag
     4741 cgatgcatgg gagctctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat
     4801 atatgtatgt tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt
     4861 tactcgttgt aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg
     4921 ctagcgatgg gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg
     4981 atctccaaaa aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt
     5041 ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa
     5101 gtaaagtaaa aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag
     5161 tactttacac ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa
     5221 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa
     5281 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     5341 aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     5401 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     5461 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     5521 actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc
     5581 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     5641 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     5701 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     5761 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     5821 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     5881 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     5941 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     6001 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt
     6061 taattggttg taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg
     6121 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6181 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6241 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6301 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6361 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6421 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6481 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6541 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6601 gttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6661 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6721 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6781 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6841 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     6901 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     6961 cagattt
//
