LOCUS       sequence-230152-        7319 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 7319)
  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 7319)
  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-eGFP
FEATURES             Location/Qualifiers
     source          1..7319
                     /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             join(3923..3925,3926..3928,3929..4642)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3923..3925,3926..3928,3929..4639)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4229..4248)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4576..4597
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4647..4871
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4941..5022
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5146..5227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5544..5562
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5585..5772,5773..5784)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5838..5857)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5849..6658
                     /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(5916..5935)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6801..7319,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7290..7309
                     /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 ccatggtgag caagggcgag gagctgttca ccggggtggt
     3961 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     4021 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     4081 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     4141 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     4201 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     4261 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     4321 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     4381 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     4441 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     4501 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     4561 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     4621 catggacgag ctgtacaagt aaagatctgt gccttctagt tgccagccat ctgttgtttg
     4681 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4741 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4801 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     4861 gggctctatg gcttctgagg cggaaagaac caaagggcga attaattcat tgatgagttt
     4921 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4981 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     5041 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     5101 tacaaatgtg gtcattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     5161 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     5221 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     5281 ggttttttaa agcaagtaaa acctctacaa atgtggtcat tcagatcaat taaccctcac
     5341 ccgggctagc gatgcatggg agctctgacg tacgttgtct agcaaggaag tgaagagcta
     5401 tccggatata tatgtatgtt atcataactg tactggctat gtcaggtggt aatgactgta
     5461 acgttacgtt actcgttgta ggcacgacat tgaatatgct cttctccgct gtggcccgaa
     5521 cgcctcccgc tagcgatggg atctaatacg actcactata gggagaccaa gggaaaatag
     5581 aatgaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg taaggagtaa
     5641 aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt gatttttaat
     5701 tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa tcaagtaaaa
     5761 ttactcaagt actttacacc tctgggatcc agctgcatca tgaacaataa aactgtctgc
     5821 ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
     5881 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     5941 cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
     6001 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     6061 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     6121 tgcatggtta ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
     6181 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     6241 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     6301 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     6361 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     6421 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     6481 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     6541 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     6601 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     6661 agaattggtt aattggttgt aacactggca aaaaggatct aggtgaagat cctttttgat
     6721 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6781 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6841 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6901 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6961 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     7021 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     7081 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     7141 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     7201 agcgccacgg ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     7261 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgt
//
