LOCUS       Exported                7559 bp ds-DNA     circular SYN 20-AUG-2021
DEFINITION  Donor for generating conditional alleles in zebrafish using precise 
            CRISPR directed genomic integration with the GeneWeld method.
ACCESSION   .
VERSION     .
KEYWORDS    pUFlip-floxed2A-KalTA4-; gcry1:BFP -1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7559)
  TITLE     Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas 
            targeted integration provide cell-type specific conditional gene 
            inactivation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7559)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7559
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     243..260
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    551..572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        587..617
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    625..641
                     /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     630..652
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     649..665
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     649..665
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             661..999
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTCHTSGRITLNN
                     ANYLKLLS"
     primer_bind     684..704
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        686..704
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(752..770)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(780..797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(780..796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(789..811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    996..1029
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    1034..1067
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     CDS             1214..1270
                     /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             3727..3747
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3751..4452
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    complement(5527..5560)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(5566..5599)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     promoter        5803..5821
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6097..6116)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6108..6917
                     /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(6175..6194)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      7060..89
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7549..9
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
       61 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      121 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      181 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
      241 gcagcgagtc agtgagcgag gaagcggaac agatttaaat ggtaccgagc agaggtgtaa
      301 aaagtactca aaaattttac tcaagtgaaa gtacaagtac ttagggaaaa ttttactcaa
      361 ttaaaagtaa aagtatctgg ctagaatctt acttgagtaa aagtaaaaaa gtactccatt
      421 aaaattgtac ttgagtatta aggaagtaaa agtaaaagca agaaagaaaa ctagagtggt
      481 ctccctttag tgagggtcaa ttgatgggag gcgttcgggc cacagcggac acgcaggtgc
      541 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
      601 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
      661 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagg
      721 acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
      781 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
      841 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
      901 ccttcccaac agttgcgcag cctgaatggc gaatggacct gccacacttc cggccgcata
      961 actttaaata atgccaatta tttaaagtta ctatcataac ttcgtatagc atacattata
     1021 cgaagttatc accataactt cgtataggat actttatacg aagttatcct aggcttacca
     1081 cccactagcg gtcagactgc agattgcagc acgaaacagg aagctgactc cacatggtca
     1141 catgctcact gaagtgttga cttccctgac agctgtgcac tttctaaacc ggttttctca
     1201 ttcatttaca gttgccacga acttctctct gttaaagcaa gcaggagacg tggaagaaaa
     1261 ccccggtcct atgaaactgc tctcatccat cgagcaagcc tgcgacattt gtcggcttaa
     1321 gaagctgaaa tgctccaagg aaaagccgaa atgtgccaaa tgcctgaaga acaattggga
     1381 atgtcgttac tctcccaaaa ccaagcgaag tccactcaca agggctcatc tgaccgaagt
     1441 ggagagcagg ctagagagac tggaacaact ctttttgctc atcttcccta gagaggacct
     1501 tgacatgatc ctcaagatgg attctctcca ggatattaaa gcccttttga ctggcttatt
     1561 cgtccaggac aatgtgaaca aagacgctgt gacagaccga ttggcaagtg tcgagaccga
     1621 tatgcctctg acactgagac agcacagaat cagcgctact tcctcaagcg aagagtcttc
     1681 taacaaggga cagagacagc tgactgtttc gagcaggtcg accccgtccc cggccgacgc
     1741 cctggacgac ggcgacctgg acatgctgcc tgctgatgct ctcgatgatt tcgatctgga
     1801 tatgctcccg gccgacgccc tggacgacta cgacctggac atcctcccgg gtaactaata
     1861 attaagtctc agccaccgtt aactgaacat gtcaaaacct gtggagactg ttgagatttg
     1921 atgttctgaa aagataaagc ctataaataa aatgttgccc aaatttcctg cctgatgttt
     1981 ttctttgtct ttgctacatg gctttgctgc tcggatcggc tcactctgtg tatgccacgt
     2041 tcactttgta ctctccttct cacggtaggt ttattatttt tagatgtgca gttagtttct
     2101 gtgaaataac acaccacaca ctgatattgt ctgtgcattg acttggtgag tgcacattgt
     2161 ttttgatctt gacatattta tatttgattg atcaggtgaa ctgtgtgaat ctaaagtgct
     2221 ccatacagat gttctgcatt gaaaatattc tcattttatt agtggaagtg agtgtatgcc
     2281 acatccaatc aatttcagca aacaccccag tatgatttaa tgcaaaaaaa tgaaggtatc
     2341 aaacacgcat tactactttg cagttaaata tttaacattt attccaacac gaaaaaaagc
     2401 agtaaataac actttgacaa acacgtcagg acatcttatt tttgtcaccc tcacaggcaa
     2461 tttagtataa tatattatat atatatatat atcatataat aatattcagt ataatatata
     2521 tatatatatc atattataat attcagtata atataaaaca caaacacata tatgtataat
     2581 ataatataac atttttattt attgagatgc ctctatggac cgtgttataa gaagtaaaga
     2641 tcaggagaag taaacatgaa gtgtaattat gaatactgat gttaaattaa gctatgatga
     2701 gttttcactg ttaatttacc atctcaatta aatgttgatg cctccatgac caagttaagc
     2761 agatgagact gaggacaact gtagaagaca agatgttcac tttgctgaat atagctggct
     2821 tgacagttat ctatgactct ataaatatat atatattttt ttttttataa aatgatttat
     2881 ttataactat atatccattt ctcagacagg tgcttcatat ccctcactcc cgtagctgtc
     2941 catgctggat ctgtccccgt tgtttttaaa aagctaaata agttattaac atgactgcat
     3001 ccagcgagcc aaacctgtct ggtgtacagc taccagagaa gcttgagatc ctagtcaccg
     3061 gcgttcctag ttctttaaac tgttacaaag tgtttggacc agcaactgaa ttgcactgta
     3121 aattaccaca tcagggtctc cggggtcaac gctgctaagc aatggactgg aaaatttgtg
     3181 acattgagtt tttggaataa ctgggaatgc tggataactc tttgggtaaa gctatggaac
     3241 aaaagtacca gcatgaacta agaaatgact gtcacagcac aacatcaaac acaatgcaag
     3301 acaaaggttc tctgcttttg ccttacttga cagaaaaagt aagctctagg ttcatcgcat
     3361 gcagacagca gcaatacatg acatatataa aggctggctc ttttctccct tccattgaac
     3421 tgaaacttcc actcagtcag acttgcgggg gatccacagc ctgactaact cgacaaaccc
     3481 ccccaaacct aaggtgagtt gatctttaag ctttttacat tttcagctcg catatatcaa
     3541 ttcgcgcgca ttgtcacact agcacccact agcggtcaga ctgcagattg cagcacgaaa
     3601 caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc tgacagctgt
     3661 gcactttcta aaccggtttt ctcattcatt tacagttcag ccggaattca cctcgagacc
     3721 atggctccaa agaagaagcg taaggtaacc atgagcgagc tgattaagga gaacatgcac
     3781 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     3841 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     3901 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     3961 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     4021 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     4081 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     4141 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct
     4201 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     4261 atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     4321 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac
     4381 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     4441 aagctcaatt aaagctgact aatttattta gcagtagata gctatattgt gtgaagtggt
     4501 aacagtccgt agatctacgg actgttacca cttcacgccg actcaaactg cgcagagaaa
     4561 cttcaaacga caacattggc atggcttttg ttatttttgg cgcttgactc aggatctaaa
     4621 aactggaacg gtgaaggtga cggcaatgtt ttttggcaaa taagcatccc cgaagttcta
     4681 caatgcatct gaggactcca tgttttgttt tctttagtca ttccaaatgt ttgttaaatg
     4741 cattgttccg aaacttattt gcctctatga aggctgccca gtaattggga gcatacttaa
     4801 cattgtagta ttgtatgtaa attatgtaac aaaacaatgt ctgggttttt gtacttttca
     4861 gccttaaaat cttgggttta tttttttttc tttgttccaa aaaaacgaag ctttaccatt
     4921 caagatgtaa aggtttccat tccccctggg catattgtaa aagctgtgtg gaacgtggcg
     4981 gtgccagaca tttggtgggg ccaacctgta cactgactaa ttcaattcca ataaaagtgc
     5041 acatgtaaga catcctaccc ctgtctcatt tcctgtcttg gctctgagtg gcttcaataa
     5101 aaatggcttt tgtgaagttg caccatgtta attacatttc agcactttca caccaggttt
     5161 ttgactaatt tgaatttatc gaagtttgac cttttttttt ttttttcttc aagctaattt
     5221 gaggttatca aaccatttca ttggaggaaa cttaatgttt aaaagcataa attcgaaaca
     5281 tgattcattt atttaactgc ttaatggtga aacaaacttc aagtaataca ccaagtgttc
     5341 cctcatatga aggtagctaa ctgcaaacgg ccttaacttt ccatcttaag ttgagctaca
     5401 taaaggcttt aacaggaagg aactgccagt ttaaaagtca aaccaccatg actgatataa
     5461 attcttgtcc atataacctt gcgtttcaca caatatagct atctactgct aaataaatta
     5521 gagctcataa cttcgtataa tgtatgctat acgaagttat gttagataac ttcgtataaa
     5581 gtatcctata cgaagttatg cgagctaact ttaaataatt ggcattattt aaagttatga
     5641 atcgataagt gaagagctat ccggatatat atgtatgtta tcataactgt actggctatg
     5701 tcaggtggta atgactgtaa cgttacgtta ctcgttgtag gcacgacatt gaatatgctc
     5761 ttctccgctg tggcccgaac gcctcccgct agcgatggga tctaatacga ctcactatag
     5821 ggagaccaag ggaaaataga atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa
     5881 ataaaattgt aaggagtaaa aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt
     5941 aaaagtattg atttttaatt gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa
     6001 gtacagtaat caagtaaaat tactcaagta ctttacacct ctgggatcca gctgcatcat
     6061 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     6121 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     6181 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     6241 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     6301 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     6361 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     6421 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     6481 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     6541 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     6601 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     6661 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     6721 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     6781 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     6841 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     6901 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcaa aaaggatcta
     6961 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7021 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7081 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7141 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7201 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7261 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7321 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7381 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7441 acagcgtgag ctatgagaaa gcgccacggt tcccgaaggg agaaaggcgg acaggtatcc
     7501 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcct
//
