LOCUS       sequence-227757-        6802 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 6802)
  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 6802)
  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-TagBFP
FEATURES             Location/Qualifiers
     source          1..6802
                     /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             3395..3415
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3419..4120
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4130..4354
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4424..4505
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4629..4710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5027..5045
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5068..5255,5256..5267)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5321..5340)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5332..6141
                     /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(5399..5418)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6284..6802,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6773..6792
                     /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 cggcattcat ccatcctttt catccctcaa atctctcatt
     2881 cacgtccccc tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca
     2941 cttgtgcagt tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga
     3001 acagaaacac tgcagacctt tatagaagaa caaatgataa gagtcctcat acataaagac
     3061 tccattagaa acgtcagtga cccaggagcc cagaccaaca gcaaagcaga cagtgaagga
     3121 tccacagcct gactaactcg acaaaccccc ccaaacctaa ggtgagttga tctttaagct
     3181 ttttacattt tcagctcgca tatatcaatt cgcgcgcatt gtcacactag cacccactag
     3241 cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca
     3301 ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta
     3361 cagttcagcc ggaattcacc tcgagaccat ggctccaaag aagaagcgta aggtaaccat
     3421 gagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa
     3481 ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat
     3541 gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag
     3601 cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa
     3661 gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt
     3721 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat
     3781 cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga
     3841 ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc
     3901 cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa
     3961 gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag
     4021 aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata
     4081 ctgcgacctc cctagcaaac tggggcacaa gctcaattaa agctgagatc tgtgccttct
     4141 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     4201 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4261 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4321 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccaaaggg
     4381 cgaattaatt cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     4441 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4501 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     4561 ttttttaaag caagtaaaac ctctacaaat gtggtcattg atgagtttgg acaaaccaca
     4621 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     4681 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     4741 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     4801 cattcagatc aattaaccct cacccgggct agcgatgcat gggagctctg acgtacgttg
     4861 tctagcaagg aagtgaagag ctatccggat atatatgtat gttatcataa ctgtactggc
     4921 tatgtcaggt ggtaatgact gtaacgttac gttactcgtt gtaggcacga cattgaatat
     4981 gctcttctcc gctgtggccc gaacgcctcc cgctagcgat gggatctaat acgactcact
     5041 atagggagac caagggaaaa tagaatgaag tgatctccaa aaaataagta ctttttgact
     5101 gtaaataaaa ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt aattaagtaa
     5161 aagtaaaagt attgattttt aattgtactc aagtaaagta aaaatcccca aaaataatac
     5221 ttaagtacag taatcaagta aaattactca agtactttac acctctggga tccagctgca
     5281 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     5341 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
     5401 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
     5461 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     5521 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     5581 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
     5641 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     5701 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     5761 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     5821 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     5881 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     5941 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     6001 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     6061 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     6121 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcaaaaagga
     6181 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6241 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6301 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6361 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6421 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6481 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6541 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6601 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6661 acctacagcg tgagctatga gaaagcgcca cggttcccga agggagaaag gcggacaggt
     6721 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6781 cctggtatct ttatagtcct gt
//
