LOCUS       sequence-251954-        7195 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 7195)
  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 7195)
  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-Cre-cmlc2-TagBFP
FEATURES             Location/Qualifiers
     source          1..7195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     225..242
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(272..283,284..446)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    533..554
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(569..574,575..592,593..599)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    607..623
                     /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     612..634
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     631..647
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     631..647
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             643..1056
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSYVQFTDRTPKFACITGRCNE"
     promoter        668..686
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(734..752)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(762..779)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(762..778)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(771..793)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             938..994
                     /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             995..2026
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             3789..3809
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3813..4514
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4524..4748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4818..4899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5023..5104
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5421..5439
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5462..5649,5650..5661)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5715..5734)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5726..6535
                     /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(5793..5812)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6678..7195,1..71)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7167..7186
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
       61 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      121 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      181 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      241 aggaagcgga acagatttaa atggtaccga gcagaggtgt aaaaagtact caaaaatttt
      301 actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct
      361 ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat
      421 taaggaagta aaagtaaaag caagaaagaa aactagagtg gtctcccttt agtgagggtc
      481 aattgatggg aggcgttcgg gccacagcgg acacgcaggt gcgcaacgca attaatgtga
      541 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
      601 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
      661 agcgcgcaat taaccctcac taaagggaac aaaagctgga ggacctcgag ggggggcccg
      721 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
      781 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
      841 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
      901 agcctgaatg gcgaatggac ctgccacact tcccggagcc acgaacttct ctctgttaaa
      961 gcaagcagga gacgtggaag aaaaccccgg tcctatgtcc aatttactga ccgtacacca
     1021 aaatttgcct gcattaccgg tcgatgcaac gagtgatgag gttcgcaaga acctgatgga
     1081 catgttcagg gatcgccagg cgttttctga gcatacctgg aaaatgcttc tgtccgtttg
     1141 ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa tggtttcccg cagaacctga
     1201 agatgttcgc gattatcttc tatatcttca ggcgcgcggt ctggcagtaa aaactatcca
     1261 gcaacatttg ggccagctaa acatgcttca tcgtcggtcc gggctgccac gaccaagtga
     1321 cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa gaaaacgttg atgccggtga
     1381 acgtgcaaaa caggctctag cgttcgaacg cactgatttc gaccaggttc gttcactcat
     1441 ggaaaatagc gatcgctgcc aggatatacg taatctggca tttctgggga ttgcttataa
     1501 caccctgtta cgtatagccg aaattgccag gatcagggtt aaagatatct cacgtactga
     1561 cggtgggaga atgttaatcc atattggcag aacgaaaacg ctggttagca ccgctggagt
     1621 agagaaggca cttagcctgg gggtaactaa actggtcgag cgatggattt ccgtctctgg
     1681 tgtagctgat gatccgaata actacctgtt ttgccgggtc agaaaaaatg gtgttgccgc
     1741 gccatctgcc accagccagc tatcaactcg cgccctggaa gggatttttg aagcaactca
     1801 tcgattgatt tacggcgcta aggatgactc tggtcagaga tacctggcct ggtctggaca
     1861 cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct ggagtttcaa taccggagat
     1921 catgcaagct ggtggctgga ccaatgtaaa tattgtcatg aactatatcc gtaacctgga
     1981 tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc gattagaact agtaattaag
     2041 tctcagccac cgttaactga acatgtcaaa acctgtggag actgttgaga tttgatgttc
     2101 tgaaaagata aagcctataa ataaaatgtt gcccaaattt cctgcctgat gtttttcttt
     2161 gtctttgcta catggctttg ctgctcggat cggctcactc tgtgtatgcc acgttcactt
     2221 tgtactctcc ttctcacggt aggtttatta tttttagatg tgcagttagt ttctgtgaaa
     2281 taacacacca cacactgata ttgtctgtgc attgacttgg tgagtgcaca ttgtttttga
     2341 tcttgacata tttatatttg attgatcagg tgaactgtgt gaatctaaag tgctccatac
     2401 agatgttctg cattgaaaat attctcattt tattagtgga agtgagtgta tgccacatcc
     2461 aatcaatttc agcaaacacc ccagtatgat ttaatgcaaa aaaatgaagg tatcaaacac
     2521 gcattactac tttgcagtta aatatttaac atttattcca acacgaaaaa aagcagtaaa
     2581 taacactttg acaaacacgt caggacatct tatttttgtc accctcacag gcaatttagt
     2641 ataatatatt atatatatat atatatcata taataatatt cagtataata tatatatata
     2701 tatcatatta taatattcag tataatataa aacacaaaca catatatgta taatataata
     2761 taacattttt atttattgag atgcctctat ggaccgtgtt ataagaagta aagatcagga
     2821 gaagtaaaca tgaagtgtaa ttatgaatac tgatgttaaa ttaagctatg atgagttttc
     2881 actgttaatt taccatctca attaaatgtt gatgcctcca tgaccaagtt aagcagatga
     2941 gactgaggac aactgtagaa gacaagatgt tcactttgct gaatatagct ggcttgacag
     3001 ttatctatga ctctataaat atatatatat ttttttttta taaaatgatt tatttataac
     3061 tatatatcca tttctcagac aggtgcttca tatccctcac tcccgtagct gtccatgctg
     3121 gatctgtccc cgttgttttt aaaaagctaa ataagttatt aacatgactg catccagcga
     3181 gccaaacctg tctggtgtac agctaccaga gaagcttgag atcctagtca ccggcggcat
     3241 tcatccatcc ttttcatccc tcaaatctct cattcacgtc cccctcccca tctgcacact
     3301 ttatctcatt ttccaccctg ctggaatctg agcacttgtg cagttatcag ggctcctgta
     3361 tttaggaggc tctgggtgtc catgtagggg acgaacagaa acactgcaga cctttataga
     3421 agaacaaatg ataagagtcc tcatacataa agactccatt agaaacgtca gtgacccagg
     3481 agcccagacc aacagcaaag cagacagtga aggatccaca gcctgactaa ctcgacaaac
     3541 ccccccaaac ctaaggtgag ttgatcttta agctttttac attttcagct cgcatatatc
     3601 aattcgcgcg cattgtcaca ctagcaccca ctagcggtca gactgcagat tgcagcacga
     3661 aacaggaagc tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct
     3721 gtgcactttc taaaccggtt ttctcattca tttacagttc agccggaatt cacctcgaga
     3781 ccatggctcc aaagaagaag cgtaaggtaa ccatgagcga gctgattaag gagaacatgc
     3841 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg
     3901 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     3961 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca
     4021 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat
     4081 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     4141 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg
     4201 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg
     4261 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc
     4321 tgatcgcaaa catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc
     4381 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct
     4441 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc
     4501 acaagctcaa ttaaagctga gatctgtgcc ttctagttgc cagccatctg ttgtttgccc
     4561 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4621 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4681 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4741 ctctatggct tctgaggcgg aaagaaccaa agggcgaatt aattcattga tgagtttgga
     4801 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4861 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4921 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4981 aaatgtggtc attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     5041 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     5101 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     5161 tttttaaagc aagtaaaacc tctacaaatg tggtcattca gatcaattaa ccctcacccg
     5221 ggctagcgat gcatgggagc tctgacgtac gttgtctagc aaggaagtga agagctatcc
     5281 ggatatatat gtatgttatc ataactgtac tggctatgtc aggtggtaat gactgtaacg
     5341 ttacgttact cgttgtaggc acgacattga atatgctctt ctccgctgtg gcccgaacgc
     5401 ctcccgctag cgatgggatc taatacgact cactataggg agaccaaggg aaaatagaat
     5461 gaagtgatct ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa
     5521 gtactttttt ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt
     5581 actcaagtaa agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta
     5641 ctcaagtact ttacacctct gggatccagc tgcatcatga acaataaaac tgtctgctta
     5701 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     5761 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5821 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     5881 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     5941 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     6001 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     6061 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     6121 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     6181 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     6241 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     6301 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     6361 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     6421 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     6481 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     6541 attggttaat tggttgtaac actggcaaaa aggatctagg tgaagatcct ttttgataat
     6601 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6661 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6721 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6781 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6841 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6901 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6961 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7021 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7081 gccacggttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7141 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctg
//
