LOCUS       sequence-229916-        6815 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 6815)
  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 6815)
  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-Fuse-cmlc2-eGFP
FEATURES             Location/Qualifiers
     source          1..6815
                     /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..687
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRRR"
     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             701..1375
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             701..1375
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(828..846)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1096..1115
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1290..1313
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3138..3158
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3162..3164,3165..3167,3168..3881)
                     /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(3162..3164,3165..3167,3168..3878)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3207..3228)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3468..3487)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3815..3836
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3886..4110
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4180..4261
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4385..4466
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4783..4801
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4824..5011,5012..5023)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5077..5096)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5088..5897
                     /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(5155..5174)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6040..6628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6529..6548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6782..6799
                     /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 cttctaggag gaggtagcgg aggtagcgga gcctcctccg aggacgtcat
      721 caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc acgagttcga
      781 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
      841 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tccagtacgg
      901 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
      961 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     1021 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     1081 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga
     1141 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc tgaagctgaa
     1201 ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga agcccgtgca
     1261 gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca acgaggacta
     1321 caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggag cctaagacta
     1381 gtaattaagt ctcagccacc gttaactgaa catgtcaaaa cctgtggaga ctgttgagat
     1441 ttgatgttct gaaaagataa agcctataaa taaaatgttg cccaaatttc ctgcctgatg
     1501 tttttctttg tctttgctac atggctttgc tgctcggatc ggctcactct gtgtatgcca
     1561 cgttcacttt gtactctcct tctcacggta ggtttattat ttttagatgt gcagttagtt
     1621 tctgtgaaat aacacaccac acactgatat tgtctgtgca ttgacttggt gagtgcacat
     1681 tgtttttgat cttgacatat ttatatttga ttgatcaggt gaactgtgtg aatctaaagt
     1741 gctccataca gatgttctgc attgaaaata ttctcatttt attagtggaa gtgagtgtat
     1801 gccacatcca atcaatttca gcaaacaccc cagtatgatt taatgcaaaa aaatgaaggt
     1861 atcaaacacg cattactact ttgcagttaa atatttaaca tttattccaa cacgaaaaaa
     1921 agcagtaaat aacactttga caaacacgtc aggacatctt atttttgtca ccctcacagg
     1981 caatttagta taatatatta tatatatata tatatcatat aataatattc agtataatat
     2041 atatatatat atcatattat aatattcagt ataatataaa acacaaacac atatatgtat
     2101 aatataatat aacattttta tttattgaga tgcctctatg gaccgtgtta taagaagtaa
     2161 agatcaggag aagtaaacat gaagtgtaat tatgaatact gatgttaaat taagctatga
     2221 tgagttttca ctgttaattt accatctcaa ttaaatgttg atgcctccat gaccaagtta
     2281 agcagatgag actgaggaca actgtagaag acaagatgtt cactttgctg aatatagctg
     2341 gcttgacagt tatctatgac tctataaata tatatatatt ttttttttat aaaatgattt
     2401 atttataact atatatccat ttctcagaca ggtgcttcat atccctcact cccgtagctg
     2461 tccatgctgg atctgtcccc gttgttttta aaaagctaaa taagttatta acatgactgc
     2521 atccagcgag ccaaacctgt ctggtgtaca gctaccagag aagcttgaga tcctagtcac
     2581 cggcggcatt catccatcct tttcatccct caaatctctc attcacgtcc ccctccccat
     2641 ctgcacactt tatctcattt tccaccctgc tggaatctga gcacttgtgc agttatcagg
     2701 gctcctgtat ttaggaggct ctgggtgtcc atgtagggga cgaacagaaa cactgcagac
     2761 ctttatagaa gaacaaatga taagagtcct catacataaa gactccatta gaaacgtcag
     2821 tgacccagga gcccagacca acagcaaagc agacagtgaa ggatccacag cctgactaac
     2881 tcgacaaacc cccccaaacc taaggtgagt tgatctttaa gctttttaca ttttcagctc
     2941 gcatatatca attcgcgcgc attgtcacac tagcacccac tagcggtcag actgcagatt
     3001 gcagcacgaa acaggaagct gactccacat ggtcacatgc tcactgaagt gttgacttcc
     3061 ctgacagctg tgcactttct aaaccggttt tctcattcat ttacagttca gccggaattc
     3121 acctcgagac catggctcca aagaagaagc gtaaggtaac catggtgagc aagggcgagg
     3181 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3241 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3301 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3361 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3421 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3481 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3541 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3601 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3661 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3721 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3781 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3841 ccgccgggat cactctcggc atggacgagc tgtacaagta aagatctgtg ccttctagtt
     3901 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3961 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     4021 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4081 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc aaagggcgaa
     4141 ttaattcatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4201 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4261 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     4321 ttaaagcaag taaaacctct acaaatgtgg tcattgatga gtttggacaa accacaacta
     4381 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     4441 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     4501 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtcatt
     4561 cagatcaatt aaccctcacc cgggctagcg atgcatggga gctctgacgt acgttgtcta
     4621 gcaaggaagt gaagagctat ccggatatat atgtatgtta tcataactgt actggctatg
     4681 tcaggtggta atgactgtaa cgttacgtta ctcgttgtag gcacgacatt gaatatgctc
     4741 ttctccgctg tggcccgaac gcctcccgct agcgatggga tctaatacga ctcactatag
     4801 ggagaccaag ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa
     4861 ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt
     4921 aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa
     4981 gtacagtaat caagtaaaat tactcaagta ctttacacct ctgggatcca gctgcatcat
     5041 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     5101 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     5161 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     5221 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     5281 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     5341 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     5401 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     5461 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     5521 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     5581 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     5641 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     5701 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     5761 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     5821 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     5881 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcaa aaaggatcta
     5941 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6001 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6061 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6121 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6181 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6241 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6301 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6361 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6421 acagcgtgag ctatgagaaa gcgccacggt tcccgaaggg agaaaggcgg acaggtatcc
     6481 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6541 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6601 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6661 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6721 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6781 cagcgagtca gtgagcgagg aagcggaaca gattt
//
