LOCUS       sequence-227763-        6796 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 6796)
  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 6796)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..6796
                     /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             3437..4114
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3437..4114
                     /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(3567..3585)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3835..3854
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4029..4052
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    4124..4348
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4418..4499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4623..4704
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5021..5039
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5062..5249,5250..5261)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5315..5334)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5326..6135
                     /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(5393..5412)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6278..6796,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6767..6786
                     /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 cctcctccga ggacgtcatc aaggagttca tgcgcttcaa
     3481 ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     3541 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
     3601 cttcgcctgg gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca
     3661 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     3721 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     3781 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     3841 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg
     3901 cgccctgaag ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc
     3961 cgaggtcaag accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac
     4021 cgacatcaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     4081 gcgcgccgag ggccgccact ccaccggcgc ctaaagctgt gatctgtgcc ttctagttgc
     4141 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     4201 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     4261 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     4321 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa agggcgaatt
     4381 aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     4441 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     4501 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     4561 aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac cacaactaga
     4621 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4681 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     4741 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtcattca
     4801 gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac gttgtctagc
     4861 aaggaagtga agagctatcc ggatatatat gtatgttatc ataactgtac tggctatgtc
     4921 aggtggtaat gactgtaacg ttacgttact cgttgtaggc acgacattga atatgctctt
     4981 ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc taatacgact cactataggg
     5041 agaccaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt gactgtaaat
     5101 aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa
     5161 aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata atacttaagt
     5221 acagtaatca agtaaaatta ctcaagtact ttacacctct gggatccagc tgcatcatga
     5281 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     5341 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     5401 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     5461 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     5521 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5581 atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc
     5641 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5701 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5761 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5821 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5881 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     5941 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     6001 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     6061 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     6121 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcaaaa aggatctagg
     6181 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6241 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6301 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6361 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6421 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6481 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6541 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6601 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6661 agcgtgagct atgagaaagc gccacggttc ccgaagggag aaaggcggac aggtatccgg
     6721 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6781 atctttatag tcctgt
//
