LOCUS       sequence-238293-        7208 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 7208)
  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 7208)
  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-eGFP
FEATURES             Location/Qualifiers
     source          1..7208
                     /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             3788..3808
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3812..3814,3815..3817,3818..4531)
                     /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(3812..3814,3815..3817,3818..4528)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4118..4137)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4465..4486
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4536..4760
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4830..4911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5035..5116
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5433..5451
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5474..5661,5662..5673)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5727..5746)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5738..6547
                     /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(5805..5824)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6690..7208,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7179..7198
                     /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 cggcggcatt
     3241 catccatcct tttcatccct caaatctctc attcacgtcc ccctccccat ctgcacactt
     3301 tatctcattt tccaccctgc tggaatctga gcacttgtgc agttatcagg gctcctgtat
     3361 ttaggaggct ctgggtgtcc atgtagggga cgaacagaaa cactgcagac ctttatagaa
     3421 gaacaaatga taagagtcct catacataaa gactccatta gaaacgtcag tgacccagga
     3481 gcccagacca acagcaaagc agacagtgaa ggatccacag cctgactaac tcgacaaacc
     3541 cccccaaacc taaggtgagt tgatctttaa gctttttaca ttttcagctc gcatatatca
     3601 attcgcgcgc attgtcacac tagcacccac tagcggtcag actgcagatt gcagcacgaa
     3661 acaggaagct gactccacat ggtcacatgc tcactgaagt gttgacttcc ctgacagctg
     3721 tgcactttct aaaccggttt tctcattcat ttacagttca gccggaattc acctcgagac
     3781 catggctcca aagaagaagc gtaaggtaac catggtgagc aagggcgagg agctgttcac
     3841 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     3901 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     3961 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4021 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4081 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4141 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4201 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4261 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4321 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4381 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4441 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4501 cactctcggc atggacgagc tgtacaagta aagatctgtg ccttctagtt gccagccatc
     4561 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     4621 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     4681 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     4741 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc aaagggcgaa ttaattcatt
     4801 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     4861 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4921 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4981 taaaacctct acaaatgtgg tcattgatga gtttggacaa accacaacta gaatgcagtg
     5041 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     5101 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     5161 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtcatt cagatcaatt
     5221 aaccctcacc cgggctagcg atgcatggga gctctgacgt acgttgtcta gcaaggaagt
     5281 gaagagctat ccggatatat atgtatgtta tcataactgt actggctatg tcaggtggta
     5341 atgactgtaa cgttacgtta ctcgttgtag gcacgacatt gaatatgctc ttctccgctg
     5401 tggcccgaac gcctcccgct agcgatggga tctaatacga ctcactatag ggagaccaag
     5461 ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt
     5521 aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg
     5581 atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat
     5641 caagtaaaat tactcaagta ctttacacct ctgggatcca gctgcatcat gaacaataaa
     5701 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac
     5761 gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg
     5821 cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc
     5881 agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt
     5941 cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac
     6001 tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt
     6061 agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg
     6121 gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc
     6181 tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg
     6241 taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc
     6301 ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa
     6361 attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc
     6421 catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa
     6481 atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt
     6541 tttctaatca gaattggtta attggttgta acactggcaa aaaggatcta ggtgaagatc
     6601 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6661 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6721 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6781 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6841 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6901 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6961 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7021 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7081 ctatgagaaa gcgccacggt tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7141 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7201 agtcctgt
//
