LOCUS       sequence-227667-        6889 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 6889)
  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 6889)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..6889
                     /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"
     CDS             3506..3526
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3530..4207
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3530..4207
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     polyA_signal    4217..4441
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4511..4592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4716..4797
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5114..5132
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5155..5342,5343..5354)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5408..5427)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5419..6228
                     /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(5486..5505)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6371..6889,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6860..6879
                     /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 tggcctcctc
     3541 cgaggacgtc atcaaggagt tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg
     3601 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
     3661 caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccctca
     3721 gttccagtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
     3781 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
     3841 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
     3901 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
     3961 ggcctccacc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagatgag
     4021 gctgaagctg aaggacggcg gccactacga cgccgaggtc aagaccacct acatggccaa
     4081 gaagcccgtg cagctgcccg gcgcctacaa gaccgacatc aagctggaca tcacctccca
     4141 caacgaggac tacaccatcg tggaacagta cgagcgcgcc gagggccgcc actccaccgg
     4201 cgcctaaagc tgtgatctgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4261 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4321 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4381 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     4441 gcttctgagg cggaaagaac caaagggcga attaattcat tgatgagttt ggacaaacca
     4501 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     4561 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     4621 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     4681 gtcattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     4741 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     4801 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     4861 agcaagtaaa acctctacaa atgtggtcat tcagatcaat taaccctcac ccgggctagc
     4921 gatgcatggg agctctgacg tacgttgtct agcaaggaag tgaagagcta tccggatata
     4981 tatgtatgtt atcataactg tactggctat gtcaggtggt aatgactgta acgttacgtt
     5041 actcgttgta ggcacgacat tgaatatgct cttctccgct gtggcccgaa cgcctcccgc
     5101 tagcgatggg atctaatacg actcactata gggagaccaa gggaaaatag aatgaagtga
     5161 tctccaaaaa ataagtactt tttgactgta aataaaattg taaggagtaa aaagtacttt
     5221 tttttctaaa aaaatgtaat taagtaaaag taaaagtatt gatttttaat tgtactcaag
     5281 taaagtaaaa atccccaaaa ataatactta agtacagtaa tcaagtaaaa ttactcaagt
     5341 actttacacc tctgggatcc agctgcatca tgaacaataa aactgtctgc ttacataaac
     5401 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat
     5461 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     5521 ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     5581 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     5641 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     5701 ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca
     5761 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     5821 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     5881 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     5941 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     6001 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     6061 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     6121 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     6181 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     6241 aattggttgt aacactggca aaaaggatct aggtgaagat cctttttgat aatctcatga
     6301 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6361 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6421 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6481 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6541 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6601 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6661 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6721 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgg
     6781 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6841 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgt
//
