LOCUS       sequence-227656-        6108 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 6108)
  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 6108)
  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-mRFP
FEATURES             Location/Qualifiers
     source          1..6108
                     /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..3426
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2749..3426
                     /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(2879..2897)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3147..3166
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3341..3364
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3436..3660
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    3730..3811
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    3935..4016
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4333..4351
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(4374..4561,4562..4573)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4627..4646)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4638..5447
                     /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(4705..4724)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(5590..6108,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6079..6098
                     /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 ggcctcctcc
     2761 gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca tggagggctc cgtgaacggc
     2821 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     2881 aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct gtcccctcag
     2941 ttccagtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     3001 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     3061 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     3121 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     3181 gcctccaccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagatgagg
     3241 ctgaagctga aggacggcgg ccactacgac gccgaggtca agaccaccta catggccaag
     3301 aagcccgtgc agctgcccgg cgcctacaag accgacatca agctggacat cacctcccac
     3361 aacgaggact acaccatcgt ggaacagtac gagcgcgccg agggccgcca ctccaccggc
     3421 gcctaaagct gtgatctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3481 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3541 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3601 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3661 cttctgaggc ggaaagaacc aaagggcgaa ttaattcatt gatgagtttg gacaaaccac
     3721 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     3781 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     3841 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     3901 tcattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     3961 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     4021 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     4081 gcaagtaaaa cctctacaaa tgtggtcatt cagatcaatt aaccctcacc cgggctagcg
     4141 atgcatggga gctctgacgt acgttgtcta gcaaggaagt gaagagctat ccggatatat
     4201 atgtatgtta tcataactgt actggctatg tcaggtggta atgactgtaa cgttacgtta
     4261 ctcgttgtag gcacgacatt gaatatgctc ttctccgctg tggcccgaac gcctcccgct
     4321 agcgatggga tctaatacga ctcactatag ggagaccaag ggaaaataga atgaagtgat
     4381 ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt
     4441 ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt
     4501 aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta
     4561 ctttacacct ctgggatcca gctgcatcat gaacaataaa actgtctgct tacataaaca
     4621 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt
     4681 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     4741 gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     4801 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     4861 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     4921 tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag
     4981 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     5041 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga
     5101 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     5161 aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg
     5221 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     5281 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     5341 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     5401 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     5461 attggttgta acactggcaa aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5521 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5581 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5641 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5701 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5761 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5821 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5881 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5941 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacggt
     6001 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6061 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgt
//
