LOCUS       sequence-237988-        6944 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 6944)
  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 6944)
  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-gal4-VP16-gcry-eGFP
FEATURES             Location/Qualifiers
     source          1..6944
                     /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..998
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSF"
     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             937..993
                     /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             1000..1437
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3524..3544
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3548..3550,3551..3553,3554..4267)
                     /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(3548..3550,3551..3553,3554..4264)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3593..3614)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3854..3873)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4201..4222
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4272..4496
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4566..4647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4771..4852
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5169..5187
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5210..5397,5398..5409)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5463..5482)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5474..6283
                     /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(5541..5560)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6426..6944,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6915..6934
                     /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 cccggagcca cgaacttctc tctgttaaag
      961 caagcaggag acgtggaaga aaaccccggt ccttctagaa agctactgtc ttctatcgaa
     1021 caagcatgcg atatttgccg acttaaaaag ctcaagtgct ccaaagaaaa accgaagtgc
     1081 gccaagtgtc tgaagaacaa ctgggagtgt cgctactctc ccaaaaccaa aaggtctccg
     1141 ctgactaggg cacatctgac agaagtggaa tcaaggctag aaagactgga acagctattt
     1201 ctactgattt ttcctcgaga agaccttgac atgattttga aaatggattc tttacaggat
     1261 ataaaagcat tgttaacagg attatttgta caagataatg tgaataaaga tgccgtcaca
     1321 gatagattgg cttcagtgga gactgatatg cctctaacat tgagacagca tagaataagt
     1381 gcgacatcat catcggaaga gagtagtaac aaaggtcaaa gacagttgac tgtatcgtcg
     1441 aggtcgaccc cgggaataca gatctctcga gccgcccccc cgaccgatgt cagcctgggg
     1501 gacgagctcc acttagacgg cgaggacgtg gcgatggcgc atgccgacgc gctagacgat
     1561 ttcgatctgg acatgttggg ggacggggat tccccgggtc cgggatttac cccccacgac
     1621 tccgccccct acggcgctct ggatatgtaa aactagtaat taagtctcag ccaccgttaa
     1681 ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa gataaagcct
     1741 ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt gctacatggc
     1801 tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact ctccttctca
     1861 cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac accacacact
     1921 gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga catatttata
     1981 tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt tctgcattga
     2041 aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa tttcagcaaa
     2101 caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta ctactttgca
     2161 gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac tttgacaaac
     2221 acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata tattatatat
     2281 atatatatat catataataa tattcagtat aatatatata tatatatcat attataatat
     2341 tcagtataat ataaaacaca aacacatata tgtataatat aatataacat ttttatttat
     2401 tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta aacatgaagt
     2461 gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt aatttaccat
     2521 ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga ggacaactgt
     2581 agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct atgactctat
     2641 aaatatatat atattttttt tttataaaat gatttattta taactatata tccatttctc
     2701 agacaggtgc ttcatatccc tcactcccgt agctgtccat gctggatctg tccccgttgt
     2761 ttttaaaaag ctaaataagt tattaacatg actgcatcca gcgagccaaa cctgtctggt
     2821 gtacagctac cagagaagct tgagatccta gtcaccggcg ttcctagttc tttaaactgt
     2881 tacaaagtgt ttggaccagc aactgaattg cactgtaaat taccacatca gggtctccgg
     2941 ggtcaacgct gctaagcaat ggactggaaa atttgtgaca ttgagttttt ggaataactg
     3001 ggaatgctgg ataactcttt gggtaaagct atggaacaaa agtaccagca tgaactaaga
     3061 aatgactgtc acagcacaac atcaaacaca atgcaagaca aaggttctct gcttttgcct
     3121 tacttgacag aaaaagtaag ctctaggttc atcgcatgca gacagcagca atacatgaca
     3181 tatataaagg ctggctcttt tctcccttcc attgaactga aacttccact cagtcagact
     3241 tgcgggggat ccacagcctg actaactcga caaacccccc caaacctaag gtgagttgat
     3301 ctttaagctt tttacatttt cagctcgcat atatcaattc gcgcgcattg tcacactagc
     3361 acccactagc ggtcagactg cagattgcag cacgaaacag gaagctgact ccacatggtc
     3421 acatgctcac tgaagtgttg acttccctga cagctgtgca ctttctaaac cggttttctc
     3481 attcatttac agttcagccg gaattcacct cgagaccatg gctccaaaga agaagcgtaa
     3541 ggtaaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     3601 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     3661 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3721 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     3781 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     3841 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     3901 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3961 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     4021 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     4081 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     4141 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     4201 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     4261 caagtaaaga tctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4321 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4381 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4441 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     4501 tgaggcggaa agaaccaaag ggcgaattaa ttcattgatg agtttggaca aaccacaact
     4561 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     4621 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     4681 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtcat
     4741 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     4801 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     4861 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     4921 gtaaaacctc tacaaatgtg gtcattcaga tcaattaacc ctcacccggg ctagcgatgc
     4981 atgggagctc tgacgtacgt tgtctagcaa ggaagtgaag agctatccgg atatatatgt
     5041 atgttatcat aactgtactg gctatgtcag gtggtaatga ctgtaacgtt acgttactcg
     5101 ttgtaggcac gacattgaat atgctcttct ccgctgtggc ccgaacgcct cccgctagcg
     5161 atgggatcta atacgactca ctatagggag accaagggaa aatagaatga agtgatctcc
     5221 aaaaaataag tactttttga ctgtaaataa aattgtaagg agtaaaaagt actttttttt
     5281 ctaaaaaaat gtaattaagt aaaagtaaaa gtattgattt ttaattgtac tcaagtaaag
     5341 taaaaatccc caaaaataat acttaagtac agtaatcaag taaaattact caagtacttt
     5401 acacctctgg gatccagctg catcatgaac aataaaactg tctgcttaca taaacagtaa
     5461 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
     5521 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     5581 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     5641 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     5701 tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac
     5761 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     5821 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     5881 ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa
     5941 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     6001 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     6061 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     6121 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     6181 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     6241 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     6301 gttgtaacac tggcaaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6361 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6421 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6481 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6541 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6601 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6661 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6721 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6781 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacggttccc
     6841 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6901 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgt
//
