LOCUS       sequence-230141-        7306 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 7306)
  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 7306)
  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-gcry-TagBFP
FEATURES             Location/Qualifiers
     source          1..7306
                     /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..1055
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRSHELLS
                     VKASRRRGRKPRSYVQFTDRTPKFACITGRCNE"
     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             994..2025
                     /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             3899..3919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3923..4624
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     polyA_signal    4634..4858
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4928..5009
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5133..5214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5531..5549
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5572..5759,5760..5771)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5825..5844)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5836..6645
                     /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(5903..5922)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6788..7306,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7277..7296
                     /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 cctatgtcca atttactgac cgtacaccaa
     1021 aatttgcctg cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac
     1081 atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc
     1141 cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa
     1201 gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag
     1261 caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac
     1321 agcaatgctg tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa
     1381 cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg
     1441 gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac
     1501 accctgttac gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac
     1561 ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac cgctggagta
     1621 gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt
     1681 gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg
     1741 ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat
     1801 cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac
     1861 agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc
     1921 atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg taacctggat
     1981 agtgaaacag gggcaatggt gcgcctgctg gaagatggcg attagaacta gtaattaagt
     2041 ctcagccacc gttaactgaa catgtcaaaa cctgtggaga ctgttgagat ttgatgttct
     2101 gaaaagataa agcctataaa taaaatgttg cccaaatttc ctgcctgatg tttttctttg
     2161 tctttgctac atggctttgc tgctcggatc ggctcactct gtgtatgcca cgttcacttt
     2221 gtactctcct tctcacggta ggtttattat ttttagatgt gcagttagtt tctgtgaaat
     2281 aacacaccac acactgatat tgtctgtgca ttgacttggt gagtgcacat tgtttttgat
     2341 cttgacatat ttatatttga ttgatcaggt gaactgtgtg aatctaaagt gctccataca
     2401 gatgttctgc attgaaaata ttctcatttt attagtggaa gtgagtgtat gccacatcca
     2461 atcaatttca gcaaacaccc cagtatgatt taatgcaaaa aaatgaaggt atcaaacacg
     2521 cattactact ttgcagttaa atatttaaca tttattccaa cacgaaaaaa agcagtaaat
     2581 aacactttga caaacacgtc aggacatctt atttttgtca ccctcacagg caatttagta
     2641 taatatatta tatatatata tatatcatat aataatattc agtataatat atatatatat
     2701 atcatattat aatattcagt ataatataaa acacaaacac atatatgtat aatataatat
     2761 aacattttta tttattgaga tgcctctatg gaccgtgtta taagaagtaa agatcaggag
     2821 aagtaaacat gaagtgtaat tatgaatact gatgttaaat taagctatga tgagttttca
     2881 ctgttaattt accatctcaa ttaaatgttg atgcctccat gaccaagtta agcagatgag
     2941 actgaggaca actgtagaag acaagatgtt cactttgctg aatatagctg gcttgacagt
     3001 tatctatgac tctataaata tatatatatt ttttttttat aaaatgattt atttataact
     3061 atatatccat ttctcagaca ggtgcttcat atccctcact cccgtagctg tccatgctgg
     3121 atctgtcccc gttgttttta aaaagctaaa taagttatta acatgactgc atccagcgag
     3181 ccaaacctgt ctggtgtaca gctaccagag aagcttgaga tcctagtcac cggcgttcct
     3241 agttctttaa actgttacaa agtgtttgga ccagcaactg aattgcactg taaattacca
     3301 catcagggtc tccggggtca acgctgctaa gcaatggact ggaaaatttg tgacattgag
     3361 tttttggaat aactgggaat gctggataac tctttgggta aagctatgga acaaaagtac
     3421 cagcatgaac taagaaatga ctgtcacagc acaacatcaa acacaatgca agacaaaggt
     3481 tctctgcttt tgccttactt gacagaaaaa gtaagctcta ggttcatcgc atgcagacag
     3541 cagcaataca tgacatatat aaaggctggc tcttttctcc cttccattga actgaaactt
     3601 ccactcagtc agacttgcgg gggatccaca gcctgactaa ctcgacaaac ccccccaaac
     3661 ctaaggtgag ttgatcttta agctttttac attttcagct cgcatatatc aattcgcgcg
     3721 cattgtcaca ctagcaccca ctagcggtca gactgcagat tgcagcacga aacaggaagc
     3781 tgactccaca tggtcacatg ctcactgaag tgttgacttc cctgacagct gtgcactttc
     3841 taaaccggtt ttctcattca tttacagttc agccggaatt cacctcgaga ccatggctcc
     3901 aaagaagaag cgtaaggtaa ccatgagcga gctgattaag gagaacatgc acatgaagct
     3961 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     4021 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     4081 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     4141 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     4201 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     4261 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     4321 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     4381 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     4441 catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     4501 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     4561 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagctcaa
     4621 ttaaagctga gatctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4681 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4741 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4801 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     4861 tctgaggcgg aaagaaccaa agggcgaatt aattcattga tgagtttgga caaaccacaa
     4921 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4981 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     5041 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggtc
     5101 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     5161 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5221 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     5281 aagtaaaacc tctacaaatg tggtcattca gatcaattaa ccctcacccg ggctagcgat
     5341 gcatgggagc tctgacgtac gttgtctagc aaggaagtga agagctatcc ggatatatat
     5401 gtatgttatc ataactgtac tggctatgtc aggtggtaat gactgtaacg ttacgttact
     5461 cgttgtaggc acgacattga atatgctctt ctccgctgtg gcccgaacgc ctcccgctag
     5521 cgatgggatc taatacgact cactataggg agaccaaggg aaaatagaat gaagtgatct
     5581 ccaaaaaata agtacttttt gactgtaaat aaaattgtaa ggagtaaaaa gtactttttt
     5641 ttctaaaaaa atgtaattaa gtaaaagtaa aagtattgat ttttaattgt actcaagtaa
     5701 agtaaaaatc cccaaaaata atacttaagt acagtaatca agtaaaatta ctcaagtact
     5761 ttacacctct gggatccagc tgcatcatga acaataaaac tgtctgctta cataaacagt
     5821 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc
     5881 aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt
     5941 gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc
     6001 aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa
     6061 tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc
     6121 accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt
     6181 gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt
     6241 aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat
     6301 aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa
     6361 gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt
     6421 gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt
     6481 ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt
     6541 gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat
     6601 atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat
     6661 tggttgtaac actggcaaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6721 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6781 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6841 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6901 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6961 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7021 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7081 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7141 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacggttc
     7201 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7261 cgagggagct tccaggggga aacgcctggt atctttatag tcctgt
//
