LOCUS       sequence-353851-        7377 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 7377)
  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 7377)
  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-; HE1A:BFP -1
FEATURES             Location/Qualifiers
     source          1..7377
                     /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..980
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSGRITLNN
                     ANYLKLLS"
     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"
     protein_bind    940..971
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(977..1010)
                     /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(1015..1048)
                     /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             1195..1251
                     /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             1252..1926
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             1252..1926
                     /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(1379..1397)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1647..1666
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1841..1864
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             3526..3546
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3550..4251
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    5326..5359
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5365..5398
                     /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    5405..5436
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        5602..5620
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(5643..5830,5831..5842)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5896..5915)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5907..6716
                     /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(5974..5993)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(6859..7377,1..70)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7348..7367
                     /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 ccggccgcat aactttaaat aatgccaatt
      961 atttaaagtt actatcataa cttcgtatag catacattat acgaagttat caccataact
     1021 tcgtatagga tactttatac gaagttatcc taggcttacc acccactagc ggtcagactg
     1081 cagattgcag cacgaaacag gaagctgact ccacatggtc acatgctcac tgaagtgttg
     1141 acttccctga cagctgtgca ctttctaaac cggttttctc attcatttac agttgccacg
     1201 aacttctctc tgttaaagca agcaggagac gtggaagaaa accccggtcc tgcctcctcc
     1261 gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca tggagggctc cgtgaacggc
     1321 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     1381 aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct gtcccctcag
     1441 ttccagtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     1501 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     1561 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     1621 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     1681 gcctccaccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagatgagg
     1741 ctgaagctga aggacggcgg ccactacgac gccgaggtca agaccaccta catggccaag
     1801 aagcccgtgc agctgcccgg cgcctacaag accgacatca agctggacat cacctcccac
     1861 aacgaggact acaccatcgt ggaacagtac gagcgcgccg agggccgcca ctccaccgga
     1921 gcctaagact agtaattaag tctcagccac cgttaactga acatgtcaaa acctgtggag
     1981 actgttgaga tttgatgttc tgaaaagata aagcctataa ataaaatgtt gcccaaattt
     2041 cctgcctgat gtttttcttt gtctttgcta catggctttg ctgctcggat cggctcactc
     2101 tgtgtatgcc acgttcactt tgtactctcc ttctcacggt aggtttatta tttttagatg
     2161 tgcagttagt ttctgtgaaa taacacacca cacactgata ttgtctgtgc attgacttgg
     2221 tgagtgcaca ttgtttttga tcttgacata tttatatttg attgatcagg tgaactgtgt
     2281 gaatctaaag tgctccatac agatgttctg cattgaaaat attctcattt tattagtgga
     2341 agtgagtgta tgccacatcc aatcaatttc agcaaacacc ccagtatgat ttaatgcaaa
     2401 aaaatgaagg tatcaaacac gcattactac tttgcagtta aatatttaac atttattcca
     2461 acacgaaaaa aagcagtaaa taacactttg acaaacacgt caggacatct tatttttgtc
     2521 accctcacag gcaatttagt ataatatatt atatatatat atatatcata taataatatt
     2581 cagtataata tatatatata tatcatatta taatattcag tataatataa aacacaaaca
     2641 catatatgta taatataata taacattttt atttattgag atgcctctat ggaccgtgtt
     2701 ataagaagta aagatcagga gaagtaaaca tgaagtgtaa ttatgaatac tgatgttaaa
     2761 ttaagctatg atgagttttc actgttaatt taccatctca attaaatgtt gatgcctcca
     2821 tgaccaagtt aagcagatga gactgaggac aactgtagaa gacaagatgt tcactttgct
     2881 gaatatagct ggcttgacag ttatctatga ctctataaat atatatatat tttttttttt
     2941 ataaaatgat ttatttataa ctatatatcc atttctcaga caggtgcttc atatccctca
     3001 ctcccgtagc tgtccatgct ggatctgtcc ccgttgtttt taaaaagcta aataagttat
     3061 taacatgact gcatccagcg agccaaacct gtctggtgta cagctaccag agaagcttga
     3121 gatcctagtc aaagctttca accactccag gcatagctag agccactgat gcatctatag
     3181 agctgaagta aagatagggt tgatgcaaat cgatttagaa ttttcaatgt agaaaaaaca
     3241 aaataaaaag tctattcact tgatggatag attagtcaaa caaacataga gtaggcactt
     3301 cccttgactc tgtaaacttt tggatttaac agattgtgca ctccttttag ttaaacacat
     3361 tagtccctga aaactaagac cctaaccata atcccattgt agacccacct actcgggtga
     3421 atataaagct cgcacagatc atctcacaga catcagacct tggcaaagga gcatcctgaa
     3481 catctctaca cactgaggca acgaattcac ctcgagacca tggctccaaa gaagaagcgt
     3541 aaggtaacca tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     3601 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     3661 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
     3721 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
     3781 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
     3841 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     3901 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
     3961 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
     4021 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca
     4081 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
     4141 agactggaaa gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca
     4201 gtggccagat actgcgacct ccctagcaaa ctggggcaca agctcaatta aagctgacta
     4261 atttatttag cagtagatag ctatattgtg tgaagtggta acagtccgta gatctacgga
     4321 ctgttaccac ttcacgccga ctcaaactgc gcagagaaac ttcaaacgac aacattggca
     4381 tggcttttgt tatttttggc gcttgactca ggatctaaaa actggaacgg tgaaggtgac
     4441 ggcaatgttt tttggcaaat aagcatcccc gaagttctac aatgcatctg aggactccat
     4501 gttttgtttt ctttagtcat tccaaatgtt tgttaaatgc attgttccga aacttatttg
     4561 cctctatgaa ggctgcccag taattgggag catacttaac attgtagtat tgtatgtaaa
     4621 ttatgtaaca aaacaatgtc tgggtttttg tacttttcag ccttaaaatc ttgggtttat
     4681 ttttttttct ttgttccaaa aaaacgaagc tttaccattc aagatgtaaa ggtttccatt
     4741 ccccctgggc atattgtaaa agctgtgtgg aacgtggcgg tgccagacat ttggtggggc
     4801 caacctgtac actgactaat tcaattccaa taaaagtgca catgtaagac atcctacccc
     4861 tgtctcattt cctgtcttgg ctctgagtgg cttcaataaa aatggctttt gtgaagttgc
     4921 accatgttaa ttacatttca gcactttcac accaggtttt tgactaattt gaatttatcg
     4981 aagtttgacc tttttttttt tttttcttca agctaatttg aggttatcaa accatttcat
     5041 tggaggaaac ttaatgttta aaagcataaa ttcgaaacat gattcattta tttaactgct
     5101 taatggtgaa acaaacttca agtaatacac caagtgttcc ctcatatgaa ggtagctaac
     5161 tgcaaacggc cttaactttc catcttaagt tgagctacat aaaggcttta acaggaagga
     5221 actgccagtt taaaagtcaa accaccatga ctgatataaa ttcttgtcca tataaccttg
     5281 cgtttcacac aatatagcta tctactgcta aataaattag agctcataac ttcgtataat
     5341 gtatgctata cgaagttatg ttagataact tcgtataaag tatcctatac gaagttatgc
     5401 gagctaactt taaataattg gcattattta aagttatgaa tcgataagtg aagagctatc
     5461 cggatatata tgtatgttat cataactgta ctggctatgt caggtggtaa tgactgtaac
     5521 gttacgttac tcgttgtagg cacgacattg aatatgctct tctccgctgt ggcccgaacg
     5581 cctcccgcta gcgatgggat ctaatacgac tcactatagg gagaccaagg gaaaatagaa
     5641 tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa taaaattgta aggagtaaaa
     5701 agtacttttt tttctaaaaa aatgtaatta agtaaaagta aaagtattga tttttaattg
     5761 tactcaagta aagtaaaaat ccccaaaaat aatacttaag tacagtaatc aagtaaaatt
     5821 actcaagtac tttacacctc tgggatccag ctgcatcatg aacaataaaa ctgtctgctt
     5881 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc
     5941 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     6001 ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc
     6061 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     6121 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     6181 catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc
     6241 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     6301 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     6361 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     6421 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     6481 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     6541 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     6601 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     6661 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     6721 aattggttaa ttggttgtaa cactggcaaa aaggatctag gtgaagatcc tttttgataa
     6781 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6841 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6901 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6961 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     7021 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7081 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7141 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7201 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7261 cgccacggtt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7321 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgt
//
