LOCUS       sequence-227760-        6778 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 6778)
  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 6778)
  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-Fuse-cmlc2-mRFP
FEATURES             Location/Qualifiers
     source          1..6778
                     /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..944
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSRRR"
     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             958..1632
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             958..1632
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             3395..3415
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3419..4096
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3419..4096
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     polyA_signal    4106..4330
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    4400..4481
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    4605..4686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5003..5021
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5044..5231,5232..5243)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5297..5316)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5308..6117
                     /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(5375..5394)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6260..6778,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6749..6768
                     /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 ctaggaggag gtagcggagg tagcggagcc
      961 tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg
     1021 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     1081 accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc
     1141 cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     1201 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     1261 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     1321 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     1381 tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     1441 atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacatg
     1501 gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct ggacatcacc
     1561 tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc
     1621 accggagcct aagactagta attaagtctc agccaccgtt aactgaacat gtcaaaacct
     1681 gtggagactg ttgagatttg atgttctgaa aagataaagc ctataaataa aatgttgccc
     1741 aaatttcctg cctgatgttt ttctttgtct ttgctacatg gctttgctgc tcggatcggc
     1801 tcactctgtg tatgccacgt tcactttgta ctctccttct cacggtaggt ttattatttt
     1861 tagatgtgca gttagtttct gtgaaataac acaccacaca ctgatattgt ctgtgcattg
     1921 acttggtgag tgcacattgt ttttgatctt gacatattta tatttgattg atcaggtgaa
     1981 ctgtgtgaat ctaaagtgct ccatacagat gttctgcatt gaaaatattc tcattttatt
     2041 agtggaagtg agtgtatgcc acatccaatc aatttcagca aacaccccag tatgatttaa
     2101 tgcaaaaaaa tgaaggtatc aaacacgcat tactactttg cagttaaata tttaacattt
     2161 attccaacac gaaaaaaagc agtaaataac actttgacaa acacgtcagg acatcttatt
     2221 tttgtcaccc tcacaggcaa tttagtataa tatattatat atatatatat atcatataat
     2281 aatattcagt ataatatata tatatatatc atattataat attcagtata atataaaaca
     2341 caaacacata tatgtataat ataatataac atttttattt attgagatgc ctctatggac
     2401 cgtgttataa gaagtaaaga tcaggagaag taaacatgaa gtgtaattat gaatactgat
     2461 gttaaattaa gctatgatga gttttcactg ttaatttacc atctcaatta aatgttgatg
     2521 cctccatgac caagttaagc agatgagact gaggacaact gtagaagaca agatgttcac
     2581 tttgctgaat atagctggct tgacagttat ctatgactct ataaatatat atatattttt
     2641 tttttataaa atgatttatt tataactata tatccatttc tcagacaggt gcttcatatc
     2701 cctcactccc gtagctgtcc atgctggatc tgtccccgtt gtttttaaaa agctaaataa
     2761 gttattaaca tgactgcatc cagcgagcca aacctgtctg gtgtacagct accagagaag
     2821 cttgagatcc tagtcaccgg cggcattcat ccatcctttt catccctcaa atctctcatt
     2881 cacgtccccc tccccatctg cacactttat ctcattttcc accctgctgg aatctgagca
     2941 cttgtgcagt tatcagggct cctgtattta ggaggctctg ggtgtccatg taggggacga
     3001 acagaaacac tgcagacctt tatagaagaa caaatgataa gagtcctcat acataaagac
     3061 tccattagaa acgtcagtga cccaggagcc cagaccaaca gcaaagcaga cagtgaagga
     3121 tccacagcct gactaactcg acaaaccccc ccaaacctaa ggtgagttga tctttaagct
     3181 ttttacattt tcagctcgca tatatcaatt cgcgcgcatt gtcacactag cacccactag
     3241 cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca
     3301 ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta
     3361 cagttcagcc ggaattcacc tcgagaccat ggctccaaag aagaagcgta aggtaaccat
     3421 ggcctcctcc gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca tggagggctc
     3481 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     3541 ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct
     3601 gtcccctcag ttccagtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     3661 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     3721 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     3781 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     3841 gggctgggag gcctccaccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     3901 caagatgagg ctgaagctga aggacggcgg ccactacgac gccgaggtca agaccaccta
     3961 catggccaag aagcccgtgc agctgcccgg cgcctacaag accgacatca agctggacat
     4021 cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgcgccg agggccgcca
     4081 ctccaccggc gcctaaagct gtgatctgtg ccttctagtt gccagccatc tgttgtttgc
     4141 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4201 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4261 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     4321 ggctctatgg cttctgaggc ggaaagaacc aaagggcgaa ttaattcatt gatgagtttg
     4381 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     4441 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     4501 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     4561 acaaatgtgg tcattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     4621 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     4681 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     4741 gttttttaaa gcaagtaaaa cctctacaaa tgtggtcatt cagatcaatt aaccctcacc
     4801 cgggctagcg atgcatggga gctctgacgt acgttgtcta gcaaggaagt gaagagctat
     4861 ccggatatat atgtatgtta tcataactgt actggctatg tcaggtggta atgactgtaa
     4921 cgttacgtta ctcgttgtag gcacgacatt gaatatgctc ttctccgctg tggcccgaac
     4981 gcctcccgct agcgatggga tctaatacga ctcactatag ggagaccaag ggaaaataga
     5041 atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa
     5101 aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt
     5161 gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat
     5221 tactcaagta ctttacacct ctgggatcca gctgcatcat gaacaataaa actgtctgct
     5281 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg
     5341 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     5401 gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt
     5461 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     5521 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     5581 gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat
     5641 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     5701 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     5761 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     5821 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     5881 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     5941 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     6001 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     6061 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca
     6121 gaattggtta attggttgta acactggcaa aaaggatcta ggtgaagatc ctttttgata
     6181 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6241 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6301 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6361 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6421 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6481 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6541 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6601 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6661 gcgccacggt tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6721 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgt
//
