LOCUS       sequence-227658-        6880 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 6880)
  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 6880)
  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-TagRFP-cmlc2-TagBFP
FEATURES             Location/Qualifiers
     source          1..6880
                     /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..1007
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSSRGV"
     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             937..993
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1000..1710
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             3473..3493
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3497..4198
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4208..4432
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4502..4583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4707..4788
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5105..5123
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5146..5333,5334..5345)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5399..5418)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5410..6219
                     /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(5477..5496)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6362..6880,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6851..6870
                     /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 cccggagcca cgaacttctc tctgttaaag
      961 caagcaggag acgtggaaga aaaccccggt cctctcgagg tgtctaaggg cgaggaactg
     1021 attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc
     1081 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     1141 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac
     1201 ggcagcagaa ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc
     1261 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     1321 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     1381 aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccaacacc
     1441 gagatgctgt accccgctga cggcggcctg gaaggcagaa gcgacatggc cctgaagctc
     1501 gtgggcgggg gccacctgat ctgcaacttc aagaccacat acagatccaa gaaacccgct
     1561 aagaacctca agatgcccgg cgtctactat gtggaccaca gactggaaag aatcaaggag
     1621 gccgacaaag agacctacgt cgagcagcac gaggtggctg tggccagata ctgcgacctc
     1681 cctagcaaac tggggcacaa acttaattga aactagtaat taagtctcag ccaccgttaa
     1741 ctgaacatgt caaaacctgt ggagactgtt gagatttgat gttctgaaaa gataaagcct
     1801 ataaataaaa tgttgcccaa atttcctgcc tgatgttttt ctttgtcttt gctacatggc
     1861 tttgctgctc ggatcggctc actctgtgta tgccacgttc actttgtact ctccttctca
     1921 cggtaggttt attattttta gatgtgcagt tagtttctgt gaaataacac accacacact
     1981 gatattgtct gtgcattgac ttggtgagtg cacattgttt ttgatcttga catatttata
     2041 tttgattgat caggtgaact gtgtgaatct aaagtgctcc atacagatgt tctgcattga
     2101 aaatattctc attttattag tggaagtgag tgtatgccac atccaatcaa tttcagcaaa
     2161 caccccagta tgatttaatg caaaaaaatg aaggtatcaa acacgcatta ctactttgca
     2221 gttaaatatt taacatttat tccaacacga aaaaaagcag taaataacac tttgacaaac
     2281 acgtcaggac atcttatttt tgtcaccctc acaggcaatt tagtataata tattatatat
     2341 atatatatat catataataa tattcagtat aatatatata tatatatcat attataatat
     2401 tcagtataat ataaaacaca aacacatata tgtataatat aatataacat ttttatttat
     2461 tgagatgcct ctatggaccg tgttataaga agtaaagatc aggagaagta aacatgaagt
     2521 gtaattatga atactgatgt taaattaagc tatgatgagt tttcactgtt aatttaccat
     2581 ctcaattaaa tgttgatgcc tccatgacca agttaagcag atgagactga ggacaactgt
     2641 agaagacaag atgttcactt tgctgaatat agctggcttg acagttatct atgactctat
     2701 aaatatatat atattttttt tttataaaat gatttattta taactatata tccatttctc
     2761 agacaggtgc ttcatatccc tcactcccgt agctgtccat gctggatctg tccccgttgt
     2821 ttttaaaaag ctaaataagt tattaacatg actgcatcca gcgagccaaa cctgtctggt
     2881 gtacagctac cagagaagct tgagatccta gtcaccggcg gcattcatcc atccttttca
     2941 tccctcaaat ctctcattca cgtccccctc cccatctgca cactttatct cattttccac
     3001 cctgctggaa tctgagcact tgtgcagtta tcagggctcc tgtatttagg aggctctggg
     3061 tgtccatgta ggggacgaac agaaacactg cagaccttta tagaagaaca aatgataaga
     3121 gtcctcatac ataaagactc cattagaaac gtcagtgacc caggagccca gaccaacagc
     3181 aaagcagaca gtgaaggatc cacagcctga ctaactcgac aaaccccccc aaacctaagg
     3241 tgagttgatc tttaagcttt ttacattttc agctcgcata tatcaattcg cgcgcattgt
     3301 cacactagca cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc
     3361 cacatggtca catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc
     3421 ggttttctca ttcatttaca gttcagccgg aattcacctc gagaccatgg ctccaaagaa
     3481 gaagcgtaag gtaaccatga gcgagctgat taaggagaac atgcacatga agctgtacat
     3541 ggagggcacc gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta
     3601 cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt
     3661 cgacatcctg gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg
     3721 catccccgac ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac
     3781 atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct
     3841 catctacaac gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa
     3901 gaaaacactc ggctgggagg ccttcaccga gacgctgtac cccgctgacg gcggcctgga
     3961 aggcagaaac gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa
     4021 gaccacatat agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt
     4081 ggactacaga ctggaaagaa tcaaggaggc caacaacgag acctacgtcg agcagcacga
     4141 ggtggcagtg gccagatact gcgacctccc tagcaaactg gggcacaagc tcaattaaag
     4201 ctgagatctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     4261 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     4321 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     4381 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     4441 gcggaaagaa ccaaagggcg aattaattca ttgatgagtt tggacaaacc acaactagaa
     4501 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     4561 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     4621 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtcattgat
     4681 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4741 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4801 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     4861 aacctctaca aatgtggtca ttcagatcaa ttaaccctca cccgggctag cgatgcatgg
     4921 gagctctgac gtacgttgtc tagcaaggaa gtgaagagct atccggatat atatgtatgt
     4981 tatcataact gtactggcta tgtcaggtgg taatgactgt aacgttacgt tactcgttgt
     5041 aggcacgaca ttgaatatgc tcttctccgc tgtggcccga acgcctcccg ctagcgatgg
     5101 gatctaatac gactcactat agggagacca agggaaaata gaatgaagtg atctccaaaa
     5161 aataagtact ttttgactgt aaataaaatt gtaaggagta aaaagtactt ttttttctaa
     5221 aaaaatgtaa ttaagtaaaa gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa
     5281 aatccccaaa aataatactt aagtacagta atcaagtaaa attactcaag tactttacac
     5341 ctctgggatc cagctgcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     5401 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     5461 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     5521 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     5581 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     5641 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     5701 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     5761 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     5821 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     5881 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     5941 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     6001 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     6061 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     6121 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     6181 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     6241 taacactggc aaaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     6541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     6601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     6661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     6721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg gttcccgaag
     6781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt
//
