LOCUS       sequence-383160-        6883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Right (RR) TALEN for mutating zebrafish rif1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6883)
  AUTHORS   Masser EA, Noble TD, Siefert JC, Goins D, Sansam CG, Sansam CL
  TITLE     Zebrafish Rif1 impacts zygotic genome activation, replication 
            timing, and sex determination
  JOURNAL   eLife 2023 (Reviewed Preprint) 12:RP87671
REFERENCE   2  (bases 1 to 6883)
  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 2023 
            (Reviewed Preprint) 12:RP87671"
COMMENT     Sequence Label: pCS2TAL3-RR-zrif1_exon8
FEATURES             Location/Qualifiers
     source          1..6883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        68..172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(173..241,242..1033)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(391..410)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1204..1792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1693..1712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1946..1963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2080..2101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2116..2121,2122..2139,2140..2146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2154..2170
                     /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     2159..2181
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2178..2194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2178..2194
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2213..2233
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2215..2233
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    2350..2484
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2387..2406)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2441..2460
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(2517..3104)
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQAREMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     CDS             complement(5208..5297)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(5298..5318)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      5321..5330
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5362..5380)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5363..5380)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(5415..6399)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      join(6470..6883,1..42)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6557..6576)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6767..6788
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
       61 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
      121 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
      181 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      241 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      301 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      361 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
      421 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
      481 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
      541 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
      601 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
      661 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
      721 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
      781 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
      841 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
      901 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
      961 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1021 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1081 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     1141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     1201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     1261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     1321 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     1381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     1441 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     1501 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     1561 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     1621 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     1681 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     1741 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     1801 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     1861 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     1921 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     1981 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     2041 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     2101 ttaggcaccc caggctttac actttatgct cccggctcgt atgttgtgtg gaattgtgag
     2161 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
     2221 ccctcactaa agggaacaaa agctgggtac gtaccgggcc caatgcattg gcgccgcggc
     2281 cgcgaattaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2341 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2401 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2461 aatgtatctt atcatgtctg gatctacgta atacgactca ctatagttct agattaaaag
     2521 tttatctcgc cgttattaaa tttccggcgc acttcctcta aggttaatgt gccggcttta
     2581 atcatttctc caccaattaa aagctcttct acactaagaa cagctccatt acaattagtg
     2641 atatgattta atcgtgtaag ctgagctttg tagtttcctt taaagtgacc actcacaaat
     2701 aaaaacttaa attccgttac agaagatgga tagactttcc accattcatt agggttgata
     2761 tgtttgtttc gtgtttgatt ttcttcgaca tatcgttgca tttcacgtgc ttggccaatt
     2821 ggcagattat aacctccgct ataagcttta gtatccacga tcacaccgta atcaatagga
     2881 gatccgacag tataaattgc tccgtccggt ttccttgatc cacccaaatg tttacctcta
     2941 tatccataaa ctttcataaa aaattccatt accttcattt caagaattct atcctgagtg
     3001 gaatttctgg caatttcaat taattcaata tattcatgag gcacatattt caatttatga
     3061 cgaagttcag atttcttctc ctccagttca cttttgacta gttgggatcc ggcaacgcga
     3121 tgggacgtgc gttcgccaat acggcgattg actcttctga tcaattccgg cgcgtgcggc
     3181 aatccctttt tcactgcatc catggcagga cgtccgccga ggcaggccaa ggcgacgagg
     3241 tggtcgttgg tcaacgcggc caacgccgga tcaggccggc tcagctgggc cacaatgctt
     3301 tcgagcgctt gcttgccgcc accgttgctg gcgatagcca ccacttggtc cggggtcagg
     3361 ccatggtcct ggcacagcac cggcaacagc cgctgcaccg tttcgagcgc ttgcttgccg
     3421 ccatcgtggc tggcgatagc caccacttgg tccggagtca ggccatggtc ctggcacagc
     3481 accggcaaca gccgctgcac cgtttcgagc gcttgcttgc cgccattgtt gctggcgata
     3541 gccaccactt ggtccggggt caggccatgg tcctggcaca gcaccggcaa cagccgctgc
     3601 accgtttcga gcgcttgctt gccgccaatg ttgctggcga tagccaccac ttggtccggg
     3661 gtcaggccat ggtcctggca cagcaccggc aacagccgct gcaccgtttc gagcgcttgc
     3721 ttgccgccat cgtggctggc gatagccacc acttggtccg gggtcaggcc atggtcctgg
     3781 cacagcaccg gcaacagccg ctgcaccgtt tcgagcgctt gcttgccgcc atcgtggctg
     3841 gcgatagcca ccacttggtc cggagtcagg ccatggtcct ggcacagcac cggcaacagc
     3901 cgctgcaccg tttcgagcgc ttgcttgccg ccatcgtggc tggcgatagc caccacttgg
     3961 tccggagtca ggccatggtc ctggcacagc accggcaaca gccgctgcac cgtttcgagc
     4021 gcttgcttgc cgccaatgtt gctggcgata gccaccactt ggtccggggt caggccatgg
     4081 tcctggcaca gcaccggcaa cagccgctgc accgtttcga gcgcttgctt gccgccaatg
     4141 ttgctggcga tagccaccac ttggtccggg gtcaggccat ggtcctggca cagcaccggc
     4201 aacagccgct gcaccgtttc gagcgcttgc ttgccgccaa tgttgctggc gatagccacc
     4261 acttggtccg gggtcaggcc atggtcctgg cacagcaccg gcaacagccg ctgcaccgtt
     4321 tcgagcgctt gcttgccgcc attgttgctg gcgatagcca ccacttggtc cggggtcagg
     4381 ccatggtcct ggcacagcac cggcaacagc cgctgcaccg tttcgagcgc ttgcttgccg
     4441 ccattgttgc tggcgatagc caccacttgg tccggggtca ggccatggtc ctggcacagc
     4501 accggcaaca gccgctgcac cgtttcgagc gcttgcttgc cgccatcgtg gctggcgata
     4561 gccaccactt ggtccggagt caggccatgg tcctggcaca gcaccggcaa cagccgctgc
     4621 accgtttcga gcgcttgctt gccgccattg ttgctggcga tagccaccac ttggtccggg
     4681 gtcaggccat ggtcctggca cagcaccggc aacagccgct gcaccgtttc gagcgcttgc
     4741 ttgccgccac cgttgctggc gatagccacc acttggtccg gggtcaggtt caggggggca
     4801 cccgtcagtg cattgcgcga tgcatgcact gcctccattg cggtcacgcc gccacgtttt
     4861 gcaatcttca caagttggcc tgtgtccaac tgtaacggcg gacctctcaa ctcccccgca
     4921 tccgtgagca aggcctccag ggcgcgtgcg ccggaccact gtttgccgac gccaacgatg
     4981 tcttcgtgtg tcgcctctgg caacgccgtg attatgtgct gatacgtgac agcgacggtc
     5041 cctaacgctg ccgggtgttg gctgagcgca acgatgtgcg cgtgtgtaaa cccatggccc
     5101 accagtgcct cgtggtgctg cgccactgtc gaacgcacct tcggtttgat cttctcttgc
     5161 tgctgctgac tgtagccgag cgtgcgtaga tccacggtac catgagcagc gtaatctgga
     5221 acgtcatatg gatacgatcc tgcatagtcc gggacgtcat agggatagcc cgcatagtca
     5281 ggaacatcgt atgggtatac cttacgcttc ttctttggag ccatggtggc agatcctgca
     5341 aaaagaacaa gtagcttgta ttctatagtg tcacctaaat caagcttgct ccgacgtccc
     5401 caggcagaat ggcggttccc taaacgagca ttgcttatat agacctccca ttaggcacgc
     5461 ctaccgccca tttacgtcaa tggaacgccc atttgcgtca ttgcccctcc ccattgacgt
     5521 caatggggat gtacttggca gccatcgcgg gccatttacc gccattgacg tcaatgggag
     5581 tactgccaat gtaccctggc gtacttccaa tagtaatgta cttgccaagt tactattaat
     5641 agatattgat gtactgccaa gtgggccatt taccgtcatt gacgtcaata gggggcgtga
     5701 gaacggatat gaatgggcaa tgagccatcc cattgacgtc aatggtgggt ggtcctattg
     5761 acgtcaatgg gcattgagcc aggcgggcca tttaccgtaa ttgacgtcaa tgggggaggc
     5821 gccatatacg tcaataggac cgcccatatg acgtcaatag gaaagaccat atatagagac
     5881 cattgacgtc aatgggggag tggctatggg cggtattagg aagccccata tatggtatat
     5941 gggaccgccc attgggaggg gctatctacg tcaataggaa aacccatata tggaatacta
     6001 tatggcatag ggccaataca tagtattgaa cctggccaat agccatattg gcatagggcc
     6061 atattggata ttgcctatat attgatcctg gcatatagcc aatatggccg ccattattgg
     6121 caccatgcca attcaatatg gcggacttgg cacagggcca agacccctcc tcaaacttgg
     6181 caccgtgcca agtagacccc tccccagttg gcacagtgcc aggtccgcca tattgaattg
     6241 gcatggtgcc aagtccgcca tattgaattg gctggggtcc agatccacca tattgaattg
     6301 gcacgagtcc atatacgcca tattgaattg gcacaggtcc agatccacca tattgaattg
     6361 gcatgagtcc atatacgcca tattgaattg gcggtcgact ggcgtaatag cgaagaggcc
     6421 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     6481 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6541 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6601 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6661 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     6721 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     6781 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     6841 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaa
//
