LOCUS       sequence-227543-        6219 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 6219)
  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 6219)
  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-Stop-gcry-mRFP
FEATURES             Location/Qualifiers
     source          1..6219
                     /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..956
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSIMIGSND
                     "
     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             2836..2856
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2860..3537
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2860..3537
                     /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(2990..3008)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3258..3277
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3452..3475
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3547..3771
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3841..3922
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4046..4127
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4444..4462
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4485..4672,4673..4684)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4738..4757)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4749..5558
                     /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(4816..4835)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(5701..6219,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6190..6209
                     /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 ctataatgat aggtagtaat gattagtaat
      961 gagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg
     1021 ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg
     1081 cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg
     1141 tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca
     1201 gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag
     1261 tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat
     1321 ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg
     1381 agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa tgcaaaaaaa
     1441 tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt attccaacac
     1501 gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt tttgtcaccc
     1561 tcacaggcaa tttagtataa tatattatat atatatatat atcatataat aatattcagt
     1621 ataatatata tatatatatc atattataat attcagtata atataaaaca caaacacata
     1681 tatgtataat ataatataac atttttattt attgagatgc ctctatggac cgtgttataa
     1741 gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat gttaaattaa
     1801 gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg cctccatgac
     1861 caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac tttgctgaat
     1921 atagctggct tgacagttat ctatgactct ataaatatat atatattttt tttttataaa
     1981 atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc cctcactccc
     2041 gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa gttattaaca
     2101 tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag cttgagatcc
     2161 tagtcaccgg cgttcctagt tctttaaact gttacaaagt gtttggacca gcaactgaat
     2221 tgcactgtaa attaccacat cagggtctcc ggggtcaacg ctgctaagca atggactgga
     2281 aaatttgtga cattgagttt ttggaataac tgggaatgct ggataactct ttgggtaaag
     2341 ctatggaaca aaagtaccag catgaactaa gaaatgactg tcacagcaca acatcaaaca
     2401 caatgcaaga caaaggttct ctgcttttgc cttacttgac agaaaaagta agctctaggt
     2461 tcatcgcatg cagacagcag caatacatga catatataaa ggctggctct tttctccctt
     2521 ccattgaact gaaacttcca ctcagtcaga cttgcggggg atccacagcc tgactaactc
     2581 gacaaacccc cccaaaccta aggtgagttg atctttaagc tttttacatt ttcagctcgc
     2641 atatatcaat tcgcgcgcat tgtcacacta gcacccacta gcggtcagac tgcagattgc
     2701 agcacgaaac aggaagctga ctccacatgg tcacatgctc actgaagtgt tgacttccct
     2761 gacagctgtg cactttctaa accggttttc tcattcattt acagttcagc cggaattcac
     2821 ctcgagacca tggctccaaa gaagaagcgt aaggtaacca tggcctcctc cgaggacgtc
     2881 atcaaggagt tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg ccacgagttc
     2941 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     3001 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccctca gttccagtac
     3061 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     3121 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     3181 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     3241 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc
     3301 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagatgag gctgaagctg
     3361 aaggacggcg gccactacga cgccgaggtc aagaccacct acatggccaa gaagcccgtg
     3421 cagctgcccg gcgcctacaa gaccgacatc aagctggaca tcacctccca caacgaggac
     3481 tacaccatcg tggaacagta cgagcgcgcc gagggccgcc actccaccgg cgcctaaagc
     3541 tgtgatctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3601 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3661 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3721 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3781 cggaaagaac caaagggcga attaattcat tgatgagttt ggacaaacca caactagaat
     3841 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     3901 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3961 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtcattgatg
     4021 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4081 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4141 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     4201 acctctacaa atgtggtcat tcagatcaat taaccctcac ccgggctagc gatgcatggg
     4261 agctctgacg tacgttgtct agcaaggaag tgaagagcta tccggatata tatgtatgtt
     4321 atcataactg tactggctat gtcaggtggt aatgactgta acgttacgtt actcgttgta
     4381 ggcacgacat tgaatatgct cttctccgct gtggcccgaa cgcctcccgc tagcgatggg
     4441 atctaatacg actcactata gggagaccaa gggaaaatag aatgaagtga tctccaaaaa
     4501 ataagtactt tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa
     4561 aaaatgtaat taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa
     4621 atccccaaaa ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc
     4681 tctgggatcc agctgcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     4741 ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg
     4801 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     4861 tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     4921 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     4981 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     5041 cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     5101 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     5161 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     5221 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     5281 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     5341 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     5401 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     5461 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     5521 aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt
     5581 aacactggca aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     5641 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5701 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5761 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5821 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5881 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5941 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6001 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6061 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgg ttcccgaagg
     6121 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6181 gcttccaggg ggaaacgcct ggtatcttta tagtcctgt
//
