LOCUS       sequence-227539-        6132 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 6132)
  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 6132)
  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-cmlc2-TagBFP
FEATURES             Location/Qualifiers
     source          1..6132
                     /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             2725..2745
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2749..3450
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    3460..3684
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3754..3835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3959..4040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4357..4375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4398..4585,4586..4597)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4651..4670)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4662..5471
                     /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(4729..4748)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(5614..6132,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6103..6122
                     /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 cggcattcat ccatcctttt catccctcaa atctctcatt cacgtccccc
     2221 tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca cttgtgcagt
     2281 tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga acagaaacac
     2341 tgcagacctt tatagaagaa caaatgataa gagtcctcat acataaagac tccattagaa
     2401 acgtcagtga cccaggagcc cagaccaaca gcaaagcaga cagtgaagga tccacagcct
     2461 gactaactcg acaaaccccc ccaaacctaa ggtgagttga tctttaagct ttttacattt
     2521 tcagctcgca tatatcaatt cgcgcgcatt gtcacactag cacccactag cggtcagact
     2581 gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca ctgaagtgtt
     2641 gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc
     2701 ggaattcacc tcgagaccat ggctccaaag aagaagcgta aggtaaccat gagcgagctg
     2761 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
     2821 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     2881 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
     2941 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
     3001 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     3061 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     3121 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
     3181 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
     3241 gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct
     3301 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
     3361 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
     3421 cctagcaaac tggggcacaa gctcaattaa agctgagatc tgtgccttct agttgccagc
     3481 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3541 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3601 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3661 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccaaaggg cgaattaatt
     3721 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     3781 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     3841 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     3901 caagtaaaac ctctacaaat gtggtcattg atgagtttgg acaaaccaca actagaatgc
     3961 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     4021 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     4081 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt cattcagatc
     4141 aattaaccct cacccgggct agcgatgcat gggagctctg acgtacgttg tctagcaagg
     4201 aagtgaagag ctatccggat atatatgtat gttatcataa ctgtactggc tatgtcaggt
     4261 ggtaatgact gtaacgttac gttactcgtt gtaggcacga cattgaatat gctcttctcc
     4321 gctgtggccc gaacgcctcc cgctagcgat gggatctaat acgactcact atagggagac
     4381 caagggaaaa tagaatgaag tgatctccaa aaaataagta ctttttgact gtaaataaaa
     4441 ttgtaaggag taaaaagtac ttttttttct aaaaaaatgt aattaagtaa aagtaaaagt
     4501 attgattttt aattgtactc aagtaaagta aaaatcccca aaaataatac ttaagtacag
     4561 taatcaagta aaattactca agtactttac acctctggga tccagctgca tcatgaacaa
     4621 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     4681 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     4741 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     4801 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     4861 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     4921 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     4981 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     5041 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     5101 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     5161 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     5221 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     5281 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     5341 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     5401 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     5461 agtttttcta atcagaattg gttaattggt tgtaacactg gcaaaaagga tctaggtgaa
     5521 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5581 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5641 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5701 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5761 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5821 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5881 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5941 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6001 tgagctatga gaaagcgcca cggttcccga agggagaaag gcggacaggt atccggtaag
     6061 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6121 ttatagtcct gt
//
