LOCUS       sequence-229889-        7004 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 7004)
  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 7004)
  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-eGFP
FEATURES             Location/Qualifiers
     source          1..7004
                     /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             join(3351..3353,3354..3356,3357..4070)
                     /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(3351..3353,3354..3356,3357..4067)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3396..3417)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3657..3676)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4004..4025
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4075..4299
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4369..4450
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4574..4655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4972..4990
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5013..5200,5201..5212)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5266..5285)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5277..6086
                     /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(5344..5363)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6229..6817
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6718..6737
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6971..6988
                     /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 atggtgagca
     3361 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     3421 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     3481 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     3541 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     3601 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     3661 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     3721 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3781 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3841 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3901 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     3961 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     4021 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agatctgtgc
     4081 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     4141 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     4201 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     4261 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     4321 aagggcgaat taattcattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     4381 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4441 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     4501 ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattgatgag tttggacaaa
     4561 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4621 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4681 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     4741 gtggtcattc agatcaatta accctcaccc gggctagcga tgcatgggag ctctgacgta
     4801 cgttgtctag caaggaagtg aagagctatc cggatatata tgtatgttat cataactgta
     4861 ctggctatgt caggtggtaa tgactgtaac gttacgttac tcgttgtagg cacgacattg
     4921 aatatgctct tctccgctgt ggcccgaacg cctcccgcta gcgatgggat ctaatacgac
     4981 tcactatagg gagaccaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt
     5041 tgactgtaaa taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta
     5101 agtaaaagta aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat
     5161 aatacttaag tacagtaatc aagtaaaatt actcaagtac tttacacctc tgggatccag
     5221 ctgcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     5281 gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat
     5341 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt
     5401 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     5461 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     5521 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggaa
     5581 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     5641 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     5701 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     5761 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     5821 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     5881 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     5941 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     6001 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     6061 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaaa
     6121 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6181 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6241 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6301 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6361 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6421 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6481 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6541 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6601 gagataccta cagcgtgagc tatgagaaag cgccacggtt cccgaaggga gaaaggcgga
     6661 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6721 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6781 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6841 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6901 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6961 gaccgagcgc agcgagtcag tgagcgagga agcggaacag attt
//
