LOCUS       sequence-383159-        6853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Left (DD) TALEN for mutating zebrafish rif1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6853)
  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 6853)
  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-DD-zrif1_exon8
FEATURES             Location/Qualifiers
     source          1..6853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        529..1513
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1548..1566
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1548..1565
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1598..1607
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1610..1630
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1631..1696
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3794..4381
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQD
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    4414..4548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4438..4457)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4492..4511
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4665..4685)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4665..4683)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4704..4720)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4704..4720)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4717..4739)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4728..4744
                     /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)."
     promoter        complement(join(4752..4758,4759..4776,4777..4782))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4797..4818
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4935..4952)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5106..5694)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5186..5205)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5865..6656,6657..6725))
                     /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     6488..6507
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6726..6830)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgaccgccaa
      541 ttcaatatgg cgtatatgga ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa
      601 ttcaatatgg cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa
      661 ttcaatatgg cggacttggc accatgccaa ttcaatatgg cggacctggc actgtgccaa
      721 ctggggaggg gtctacttgg cacggtgcca agtttgagga ggggtcttgg ccctgtgcca
      781 agtccgccat attgaattgg catggtgcca ataatggcgg ccatattggc tatatgccag
      841 gatcaatata taggcaatat ccaatatggc cctatgccaa tatggctatt ggccaggttc
      901 aatactatgt attggcccta tgccatatag tattccatat atgggttttc ctattgacgt
      961 agatagcccc tcccaatggg cggtcccata taccatatat ggggcttcct aataccgccc
     1021 atagccactc ccccattgac gtcaatggtc tctatatatg gtctttccta ttgacgtcat
     1081 atgggcggtc ctattgacgt atatggcgcc tcccccattg acgtcaatta cggtaaatgg
     1141 cccgcctggc tcaatgccca ttgacgtcaa taggaccacc caccattgac gtcaatggga
     1201 tggctcattg cccattcata tccgttctca cgccccctat tgacgtcaat gacggtaaat
     1261 ggcccacttg gcagtacatc aatatctatt aatagtaact tggcaagtac attactattg
     1321 gaagtacgcc agggtacatt ggcagtactc ccattgacgt caatggcggt aaatggcccg
     1381 cgatggctgc caagtacatc cccattgacg tcaatgggga ggggcaatga cgcaaatggg
     1441 cgttccattg acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata taagcaatgc
     1501 tcgtttaggg aaccgccatt ctgcctgggg acgtcggagc aagcttgatt taggtgacac
     1561 tatagaatac aagctacttg ttctttttgc aggatctgcc accatggctc caaagaagaa
     1621 gcgtaaggta gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa
     1681 ggatgacgat gacaagggta ccgtggatct acgcacgctc ggctacagtc agcagcagca
     1741 agagaagatc aaaccgaagg tgcgttcgac agtggcgcag caccacgagg cactggtggg
     1801 ccatgggttt acacacgcgc acatcgttgc gctcagccaa cacccggcag cgttagggac
     1861 cgtcgctgtc acgtatcagc acataatcac ggcgttgcca gaggcgacac acgaagacat
     1921 cgttggcgtc ggcaaacagt ggtccggcgc acgcgccctg gaggccttgc tcacggatgc
     1981 gggggagttg agaggtccgc cgttacagtt ggacacaggc caacttgtga agattgcaaa
     2041 acgtggcggc gtgaccgcaa tggaggcagt gcatgcatcg cgcaatgcac tgacgggtgc
     2101 ccccctgaac ctgaccccgg accaagtggt ggctatcgcc agcaacaatg gcggcaagca
     2161 agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc
     2221 ggaccaagtg gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca
     2281 gcggctgttg ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat
     2341 cgccagcaac ggtggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct
     2401 gtgccaggac catggcctga ccccggacca agtggtggct atcgccagca acggtggcgg
     2461 caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct
     2521 gaccccggac caagtggtgg ctatcgccag caacggtggc ggcaagcaag cgctcgaaac
     2581 ggtgcagcgg ctgttgccgg tgctgtgcca ggaccatggc ctgactccgg accaagtggt
     2641 ggctatcgcc agccacgatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc
     2701 ggtgctgtgc caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacat
     2761 tggcggcaag caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca
     2821 tggcctgacc ccggaccaag tggtggctat cgccagcaac ggtggcggca agcaagcgct
     2881 cgaaacggtg cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca
     2941 agtggtggct atcgccagca acggtggcgg caagcaagcg ctcgaaacgg tgcagcggct
     3001 gttgccggtg ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag
     3061 caacattggc ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca
     3121 ggaccatggc ctgaccccgg accaagtggt ggctatcgcc agcaacattg gcggcaagca
     3181 agcgctcgaa acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc
     3241 ggaccaagtg gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca
     3301 gcggctgttg ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat
     3361 cgccagcaac ggtggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct
     3421 gtgccaggac catggcctga ctccggacca agtggtggct atcgccagcc acgatggcgg
     3481 caagcaagcg ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct
     3541 gaccccggac caagtggtgg ctatcgccag ccacgatggc ggcaagcaag cgctcgaaag
     3601 cattgtggcc cagctgagcc ggcctgatcc ggcgttggcc gcgttgacca acgaccacct
     3661 cgtcgccttg gcctgcctcg gcggacgtcc tgccatggat gcagtgaaaa agggattgcc
     3721 gcacgcgccg gaattgatca gaagagtcaa tcgccgtatt ggcgaacgca cgtcccatcg
     3781 cgttgccgga tcccaactag tcaaaagtga actggaggag aagaaatctg aacttcgtca
     3841 taaattgaaa tatgtgcctc atgaatatat tgaattaatt gaaattgcca gaaattccac
     3901 tcaggataga attcttgaaa tgaaggtaat ggaatttttt atgaaagttt atggatatag
     3961 aggtaaacat ttgggtggat caaggaaacc ggacggagca atttatactg tcggatctcc
     4021 tattgattac ggtgtgatcg tggatactaa agcttatagc ggaggttata atctgccaat
     4081 tggccaagca gatgaaatgc aagactatgt cgaagaaaat caaacacgaa acaaacatat
     4141 caaccctaat gaatggtgga aagtctatcc atcttctgta acggaattta agtttttatt
     4201 tgtgagtggt cactttaaag gaaactacaa agctcagctt acacgattaa atcatatcac
     4261 taattgtaat ggagctgttc ttagtgtaga agagctttta attggtggag aaatgattaa
     4321 agccggcaca ttaaccttag aggaagtgcg ccggaaattt aataacggcg agataaactt
     4381 ttaatctaga actatagtga gtcgtattac gtagatccag acatgataag atacattgat
     4441 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     4501 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     4561 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc
     4621 gcggcgccaa tgcattgggc ccggtacgta cccagctttt gttcccttta gtgagggtta
     4681 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4741 acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga
     4801 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4861 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4921 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4981 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5041 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5101 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5161 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5221 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5281 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5341 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5401 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5461 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5521 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     5581 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5641 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5701 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5761 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5821 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5881 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5941 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6001 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6061 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6121 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6181 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6241 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6301 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6361 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6421 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6481 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6541 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6601 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6661 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6721 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6781 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6841 cgaaaagtgc cac
//
