LOCUS       sequence-340513-        7651 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor for generating conditional alleles in zebrafish using precise 
            CRISPR directed genomic integration with the GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7651)
  AUTHORS   Liu F, Kambakam S, Almeida MP, Ming Z, Welker JM, Wierson WA, 
            Schultz-Rogers LE, Ekker SC, Clark KJ, Essner JJ, McGrail M
  TITLE     Cre/lox regulated conditional rescue and inactivation with zebrafish
            UFlip alleles generated by CRISPR-Cas9 targeted integration.
  JOURNAL   Elife. 2022 Jun 17;11. pii: 71478. doi: 10.7554/eLife.71478.
  PUBMED    35713402
REFERENCE   2  (bases 1 to 7651)
  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: "2022-06-17"; volume: "11. pii: 71478. doi"; pages: " 
            10.7554/eLife.71478"
COMMENT     Sequence Label: pUFlip-floxed2A-mRFP-; gcry1:BFP -2
FEATURES             Location/Qualifiers
     source          1..7651
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     295..312
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    join(342..353,354..516)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    603..624
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(639..644,645..662,663..669)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    677..693
                     /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     682..704
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     701..717
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     701..717
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             713..1051
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSGRITLNN
                     ANYLKLLS"
     promoter        738..756
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(804..822)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(832..849)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(832..848)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(841..863)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    1011..1042
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(1048..1081)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1086..1119)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             1265..1321
                     /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             1322..1996
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1322..1996
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(1449..1467)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1717..1736
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1911..1934
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3871..3891
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3895..4596
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5671..5704
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5710..5743
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    5750..5781
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5947..5965
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5988..6175,6176..6187)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6241..6260)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6252..7061
                     /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(6319..6338)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(7204..7651,1..141)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
       61 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
      121 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
      181 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
      241 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
      301 tcagtgagcg aggaagcgga acagatttaa atggtaccga gcagaggtgt aaaaagtact
      361 caaaaatttt actcaagtga aagtacaagt acttagggaa aattttactc aattaaaagt
      421 aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt
      481 acttgagtat taaggaagta aaagtaaaag caagaaagaa aactagagtg gtctcccttt
      541 agtgagggtc aattgatggg aggcgttcgg gccacagcgg acacgcaggt gcgcaacgca
      601 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      661 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      721 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga ggacctcgag
      781 ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt
      841 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
      901 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
      961 acagttgcgc agcctgaatg gcgaatggac ctgccacact tccggccgca taactttaaa
     1021 taatgccaat tatttaaagt tactatcata acttcgtata gcatacatta tacgaagtta
     1081 tcaccataac ttcgtatagg atactttata cgaagttatc ctaggcttac cacccactag
     1141 cggtcagact gcagattgca gcacgaaaca ggaagctgac tccacatggt cacatgctca
     1201 ctgaagtgtt gacttccctg acagctgtgc actttctaaa ccggttttct cattcattta
     1261 cagcgccacg aacttctctc tgttaaagca agcaggagac gtggaagaaa accccggtcc
     1321 tgcctcctcc gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca tggagggctc
     1381 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1441 ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct
     1501 gtcccctcag ttccagtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     1561 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1621 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     1681 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat
     1741 gggctgggag gcctccaccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     1801 caagatgagg ctgaagctga aggacggcgg ccactacgac gccgaggtca agaccaccta
     1861 catggccaag aagcccgtgc agctgcccgg cgcctacaag accgacatca agctggacat
     1921 cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgcgccg agggccgcca
     1981 ctccaccgga gcctaagact agtaattaag tctcagccac cgttaactga acatgtcaaa
     2041 acctgtggag actgttgaga tttgatgttc tgaaaagata aagcctataa ataaaatgtt
     2101 gcccaaattt cctgcctgat gtttttcttt gtctttgcta catggctttg ctgctcggat
     2161 cggctcactc tgtgtatgcc acgttcactt tgtactctcc ttctcacggt aggtttatta
     2221 tttttagatg tgcagttagt ttctgtgaaa taacacacca cacactgata ttgtctgtgc
     2281 attgacttgg tgagtgcaca ttgtttttga tcttgacata tttatatttg attgatcagg
     2341 tgaactgtgt gaatctaaag tgctccatac agatgttctg cattgaaaat attctcattt
     2401 tattagtgga agtgagtgta tgccacatcc aatcaatttc agcaaacacc ccagtatgat
     2461 ttaatgcaaa aaaatgaagg tatcaaacac gcattactac tttgcagtta aatatttaac
     2521 atttattcca acacgaaaaa aagcagtaaa taacactttg acaaacacgt caggacatct
     2581 tatttttgtc accctcacag gcaatttagt ataatatatt atatatatat atatatcata
     2641 taataatatt cagtataata tatatatata tatcatatta taatattcag tataatataa
     2701 aacacaaaca catatatgta taatataata taacattttt atttattgag atgcctctat
     2761 ggaccgtgtt ataagaagta aagatcagga gaagtaaaca tgaagtgtaa ttatgaatac
     2821 tgatgttaaa ttaagctatg atgagttttc actgttaatt taccatctca attaaatgtt
     2881 gatgcctcca tgaccaagtt aagcagatga gactgaggac aactgtagaa gacaagatgt
     2941 tcactttgct gaatatagct ggcttgacag ttatctatga ctctataaat atatatatat
     3001 tttttttttt ataaaatgat ttatttataa ctatatatcc atttctcaga caggtgcttc
     3061 atatccctca ctcccgtagc tgtccatgct ggatctgtcc ccgttgtttt taaaaagcta
     3121 aataagttat taacatgact gcatccagcg agccaaacct gtctggtgta cagctaccag
     3181 agaagcttga gatcctagtc accggcgttc ctagttcttt aaactgttac aaagtgtttg
     3241 gaccagcaac tgaattgcac tgtaaattac cacatcaggg tctccggggt caacgctgct
     3301 aagcaatgga ctggaaaatt tgtgacattg agtttttgga ataactggga atgctggata
     3361 actctttggg taaagctatg gaacaaaagt accagcatga actaagaaat gactgtcaca
     3421 gcacaacatc aaacacaatg caagacaaag gttctctgct tttgccttac ttgacagaaa
     3481 aagtaagctc taggttcatc gcatgcagac agcagcaata catgacatat ataaaggctg
     3541 gctcttttct cccttccatt gaactgaaac ttccactcag tcagacttgc gggggatcca
     3601 cagcctgact aactcgacaa acccccccaa acctaaggtg agttgatctt taagcttttt
     3661 acattttcag ctcgcatata tcaattcgcg cgcattgtca cactagcacc cactagcggt
     3721 cagactgcag attgcagcac gaaacaggaa gctgactcca catggtcaca tgctcactga
     3781 agtgttgact tccctgacag ctgtgcactt tctaaaccgg ttttctcatt catttacagt
     3841 tcagccggaa ttcacctcga gaccatggct ccaaagaaga agcgtaaggt aaccatgagc
     3901 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     3961 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     4021 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     4081 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     4141 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     4201 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     4261 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     4321 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     4381 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     4441 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     4501 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     4561 gacctcccta gcaaactggg gcacaagctc aattaaagct gactaattta tttagcagta
     4621 gatagctata ttgtgtgaag tggtaacagt ccgtagatct acggactgtt accacttcac
     4681 gccgactcaa actgcgcaga gaaacttcaa acgacaacat tggcatggct tttgttattt
     4741 ttggcgcttg actcaggatc taaaaactgg aacggtgaag gtgacggcaa tgttttttgg
     4801 caaataagca tccccgaagt tctacaatgc atctgaggac tccatgtttt gttttcttta
     4861 gtcattccaa atgtttgtta aatgcattgt tccgaaactt atttgcctct atgaaggctg
     4921 cccagtaatt gggagcatac ttaacattgt agtattgtat gtaaattatg taacaaaaca
     4981 atgtctgggt ttttgtactt ttcagcctta aaatcttggg tttatttttt tttctttgtt
     5041 ccaaaaaaac gaagctttac cattcaagat gtaaaggttt ccattccccc tgggcatatt
     5101 gtaaaagctg tgtggaacgt ggcggtgcca gacatttggt ggggccaacc tgtacactga
     5161 ctaattcaat tccaataaaa gtgcacatgt aagacatcct acccctgtct catttcctgt
     5221 cttggctctg agtggcttca ataaaaatgg cttttgtgaa gttgcaccat gttaattaca
     5281 tttcagcact ttcacaccag gtttttgact aatttgaatt tatcgaagtt tgaccttttt
     5341 tttttttttt cttcaagcta atttgaggtt atcaaaccat ttcattggag gaaacttaat
     5401 gtttaaaagc ataaattcga aacatgattc atttatttaa ctgcttaatg gtgaaacaaa
     5461 cttcaagtaa tacaccaagt gttccctcat atgaaggtag ctaactgcaa acggccttaa
     5521 ctttccatct taagttgagc tacataaagg ctttaacagg aaggaactgc cagtttaaaa
     5581 gtcaaaccac catgactgat ataaattctt gtccatataa ccttgcgttt cacacaatat
     5641 agctatctac tgctaaataa attagagctc ataacttcgt ataatgtatg ctatacgaag
     5701 ttatgttaga taacttcgta taaagtatcc tatacgaagt tatgcgagct aactttaaat
     5761 aattggcatt atttaaagtt atgaatcgat aagtgaagag ctatccggat atatatgtat
     5821 gttatcataa ctgtactggc tatgtcaggt ggtaatgact gtaacgttac gttactcgtt
     5881 gtaggcacga cattgaatat gctcttctcc gctgtggccc gaacgcctcc cgctagcgat
     5941 gggatctaat acgactcact atagggagac caagggaaaa tagaatgaag tgatctccaa
     6001 aaaataagta ctttttgact gtaaataaaa ttgtaaggag taaaaagtac ttttttttct
     6061 aaaaaaatgt aattaagtaa aagtaaaagt attgattttt aattgtactc aagtaaagta
     6121 aaaatcccca aaaataatac ttaagtacag taatcaagta aaattactca agtactttac
     6181 acctctggga tccagctgca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     6241 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     6301 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     6361 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     6421 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     6481 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     6541 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     6601 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     6661 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     6721 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     6781 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     6841 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     6901 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     6961 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     7021 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     7081 tgtaacactg gcaaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7321 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     7381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7441 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7501 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7561 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cggttcccga
     7621 agggagaaag gcggacaggt atccggtaag c
//
