LOCUS       sequence-227666-        6926 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 6926)
  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 6926)
  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-gcry-eGFP
FEATURES             Location/Qualifiers
     source          1..6926
                     /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..944
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRRR"
     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             958..1632
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             958..1632
                     /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(1085..1103)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1353..1372
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1547..1570
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3506..3526
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3530..3532,3533..3535,3536..4249)
                     /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(3530..3532,3533..3535,3536..4246)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3575..3596)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3836..3855)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4183..4204
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4254..4478
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4548..4629
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4753..4834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5151..5169
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5192..5379,5380..5391)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5445..5464)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5456..6265
                     /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(5523..5542)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6408..6926,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6897..6916
                     /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 ctaggaggag gtagcggagg tagcggagcc
      961 tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg
     1021 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1081 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1141 cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1201 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1261 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1321 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1381 tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1441 atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacatg
     1501 gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct ggacatcacc
     1561 tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc
     1621 accggagcct aagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct
     1681 gtggagactg ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc
     1741 aaatttcctg cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc
     1801 tcactctgtg tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt
     1861 tagatgtgca gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg
     1921 acttggtgag tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa
     1981 ctgtgtgaat ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt
     2041 agtggaagtg agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa
     2101 tgcaaaaaaa tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt
     2161 attccaacac gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt
     2221 tttgtcaccc tcacaggcaa tttagtataa tatattatat atatatatat atcatataat
     2281 aatattcagt ataatatata tatatatatc atattataat attcagtata atataaaaca
     2341 caaacacata tatgtataat ataatataac atttttattt attgagatgc ctctatggac
     2401 cgtgttataa gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat
     2461 gttaaattaa gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg
     2521 cctccatgac caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac
     2581 tttgctgaat atagctggct tgacagttat ctatgactct ataaatatat atatattttt
     2641 tttttataaa atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc
     2701 cctcactccc gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa
     2761 gttattaaca tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag
     2821 cttgagatcc tagtcaccgg cgttcctagt tctttaaact gttacaaagt gtttggacca
     2881 gcaactgaat tgcactgtaa attaccacat cagggtctcc ggggtcaacg ctgctaagca
     2941 atggactgga aaatttgtga cattgagttt ttggaataac tgggaatgct ggataactct
     3001 ttgggtaaag ctatggaaca aaagtaccag catgaactaa gaaatgactg tcacagcaca
     3061 acatcaaaca caatgcaaga caaaggttct ctgcttttgc cttacttgac agaaaaagta
     3121 agctctaggt tcatcgcatg cagacagcag caatacatga catatataaa ggctggctct
     3181 tttctccctt ccattgaact gaaacttcca ctcagtcaga cttgcggggg atccacagcc
     3241 tgactaactc gacaaacccc cccaaaccta aggtgagttg atctttaagc tttttacatt
     3301 ttcagctcgc atatatcaat tcgcgcgcat tgtcacacta gcacccacta gcggtcagac
     3361 tgcagattgc agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt
     3421 tgacttccct gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc
     3481 cggaattcac ctcgagacca tggctccaaa gaagaagcgt aaggtaacca tggtgagcaa
     3541 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3601 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3661 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3721 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     3781 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3841 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3901 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3961 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     4021 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     4081 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     4141 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     4201 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatctgtgcc
     4261 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4321 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4381 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4441 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccaa
     4501 agggcgaatt aattcattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4561 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4621 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     4681 gaggtttttt aaagcaagta aaacctctac aaatgtggtc attgatgagt ttggacaaac
     4741 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     4801 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     4861 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     4921 tggtcattca gatcaattaa ccctcacccg ggctagcgat gcatgggagc tctgacgtac
     4981 gttgtctagc aaggaagtga agagctatcc ggatatatat gtatgttatc ataactgtac
     5041 tggctatgtc aggtggtaat gactgtaacg ttacgttact cgttgtaggc acgacattga
     5101 atatgctctt ctccgctgtg gcccgaacgc ctcccgctag cgatgggatc taatacgact
     5161 cactataggg agaccaaggg aaaatagaat gaagtgatct ccaaaaaata agtacttttt
     5221 gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt ttctaaaaaa atgtaattaa
     5281 gtaaaagtaa aagtattgat ttttaattgt actcaagtaa agtaaaaatc cccaaaaata
     5341 atacttaagt acagtaatca agtaaaatta ctcaagtact ttacacctct gggatccagc
     5401 tgcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     5461 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     5521 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     5581 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     5641 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     5701 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     5761 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     5821 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     5881 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     5941 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     6001 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     6061 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     6121 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     6181 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     6241 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcaaaa
     6301 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6361 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6421 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6481 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6541 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6601 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6661 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6721 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6781 agatacctac agcgtgagct atgagaaagc gccacggttc ccgaagggag aaaggcggac
     6841 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6901 aacgcctggt atctttatag tcctgt
//
