LOCUS       sequence-227661-        6833 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 6833)
  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 6833)
  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-cmlc2-eGFP
FEATURES             Location/Qualifiers
     source          1..6833
                     /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             3413..3433
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3437..3439,3440..3442,3443..4156)
                     /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(3437..3439,3440..3442,3443..4153)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3482..3503)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3743..3762)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4090..4111
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4161..4385
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4455..4536
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4660..4741
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5058..5076
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5099..5286,5287..5298)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5352..5371)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5363..6172
                     /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(5430..5449)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6315..6833,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6804..6823
                     /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 gcattcatcc atccttttca
     2881 tccctcaaat ctctcattca cgtccccctc cccatctgca cactttatct cattttccac
     2941 cctgctggaa tctgagcact tgtgcagtta tcagggctcc tgtatttagg aggctctggg
     3001 tgtccatgta ggggacgaac agaaacactg cagaccttta tagaagaaca aatgataaga
     3061 gtcctcatac ataaagactc cattagaaac gtcagtgacc caggagccca gaccaacagc
     3121 aaagcagaca gtgaaggatc cacagcctga ctaactcgac aaaccccccc aaacctaagg
     3181 tgagttgatc tttaagcttt ttacattttc agctcgcata tatcaattcg cgcgcattgt
     3241 cacactagca cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc
     3301 cacatggtca catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc
     3361 ggttttctca ttcatttaca gttcagccgg aattcacctc gagaccatgg ctccaaagaa
     3421 gaagcgtaag gtaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3481 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3541 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3601 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     3661 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3721 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3781 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3841 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3901 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3961 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     4021 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     4081 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     4141 cgagctgtac aagtaaagat ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4201 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4261 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4321 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     4381 tatggcttct gaggcggaaa gaaccaaagg gcgaattaat tcattgatga gtttggacaa
     4441 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct
     4501 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt
     4561 atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa
     4621 tgtggtcatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4681 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4741 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     4801 ttaaagcaag taaaacctct acaaatgtgg tcattcagat caattaaccc tcacccgggc
     4861 tagcgatgca tgggagctct gacgtacgtt gtctagcaag gaagtgaaga gctatccgga
     4921 tatatatgta tgttatcata actgtactgg ctatgtcagg tggtaatgac tgtaacgtta
     4981 cgttactcgt tgtaggcacg acattgaata tgctcttctc cgctgtggcc cgaacgcctc
     5041 ccgctagcga tgggatctaa tacgactcac tatagggaga ccaagggaaa atagaatgaa
     5101 gtgatctcca aaaaataagt actttttgac tgtaaataaa attgtaagga gtaaaaagta
     5161 cttttttttc taaaaaaatg taattaagta aaagtaaaag tattgatttt taattgtact
     5221 caagtaaagt aaaaatcccc aaaaataata cttaagtaca gtaatcaagt aaaattactc
     5281 aagtacttta cacctctggg atccagctgc atcatgaaca ataaaactgt ctgcttacat
     5341 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt
     5401 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     5461 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     5521 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     5581 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     5641 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga
     5701 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     5761 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     5821 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     5881 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     5941 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     6001 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     6061 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     6121 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     6181 ggttaattgg ttgtaacact ggcaaaaagg atctaggtga agatcctttt tgataatctc
     6241 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6301 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6361 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6421 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6481 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6541 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6601 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6661 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6721 acggttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6781 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgt
//
