LOCUS       sequence-205270-        7217 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid to carry out IVT of AsCpf1 (zebrafish codon optimized).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7217)
  AUTHORS   Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, 
            Khokha MK, Doudna JA, Giraldez AJ
  TITLE     CRISPR-Cpf1 mediates efficient homology-directed repair and 
            temperature-controlled genome editing.
  JOURNAL   Nat Commun. 2017 Dec 8;8(1):2024. doi: 10.1038/s41467-017-01836-2.
  PUBMED    29222508
REFERENCE   2  (bases 1 to 7217)
  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: "Nat 
            Commun."; date: "2017-12-8"; volume: "8(1):2024. doi"; pages: " 
            10.1038/s41467-017-01836-2"
COMMENT     Sequence Label: pT3TS-zAsCpf1
FEATURES             Location/Qualifiers
     source          1..7217
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           44..86
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      102..111
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             108..4031
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="MGTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARN
                     DHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYR
                     NAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSF
                     DKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREH
                     FENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEV
                     LNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTL
                     LRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELT
                     GKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTT
                     LKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNK
                     ARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRY
                     KALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIE
                     PLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSI
                     DLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAK
                     GHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKL
                     KDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFF
                     FHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQ
                     RSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQA
                     VVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTD
                     QFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHY
                     DVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIE
                     NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQ
                     MRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKE
                     SKDLKLQNGISNQDWLAYIQELRN"
     CDS             4032..4079
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4086..4166
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     3'UTR           4194..4319
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(4405..4424)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4406..4424)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4431..4448)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4431..4447)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4440..4462)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4589..5044)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4726..4747)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4938..4957
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5071..5175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5176..5244,5245..6036)
                     /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(5394..5413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6207..6795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6696..6715
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6949..6966
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7083..7104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7119..7124,7125..7142,7143..7149)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7157..7173
                     /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     7162..7184
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7181..7197
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7181..7197
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcaga tctaactcga cgccaccatg ggcacccagt
      121 tcgagggatt caccaacctg taccaggtgt ccaagaccct gagattcgag ctgatccctc
      181 agggaaagac cctgaagcac atccaggaac agggattcat cgaagaggac aaggccagaa
      241 acgaccacta caaagagctg aagcccatca tcgaccgcat ctacaagacc tacgccgacc
      301 agtgcctgca gctcgtgcag ctggactggg agaacctgag cgccgccatc gacagctaca
      361 gaaaagagaa aaccgaggaa acacgcaacg ccctgatcga agagcaggcc acctacagaa
      421 acgccatcca cgactacttc atcggaagaa ccgacaacct gaccgacgcc atcaacaaga
      481 gacacgccga gatctacaag ggactgttca aggccgagct gttcaacgga aaggtgctga
      541 agcagctggg aaccgtgacc accaccgagc acgagaacgc cctgctgaga agcttcgaca
      601 agttcaccac ctacttcagc ggcttctacg agaacagaaa gaacgtgttc agcgccgagg
      661 acatcagcac cgccatccct cacagaatcg tgcaggacaa cttccccaag ttcaaagaga
      721 actgccacat cttcaccaga ctgatcaccg ccgtgcctag cctgcgcgag cacttcgaga
      781 acgtgaagaa ggccatcgga atcttcgtgt ctacctctat cgaggaagtg ttcagcttcc
      841 cattctacaa ccagctgctg acccagaccc agatcgacct gtataatcag ctgctgggag
      901 gaatcagcag agaggccgga accgagaaga tcaagggact gaacgaggtg ctgaacctgg
      961 ccatccagaa gaacgacgag acagcccaca tcattgccag cctgcctcac agattcatcc
     1021 ctctgttcaa gcagatcctg agcgacagaa acaccctgag cttcatcctg gaagagttca
     1081 agagcgacga ggaagtgatc cagagcttct gcaagtacaa gacactgctg agaaacgaga
     1141 acgtgctgga gacagccgag gccctgttca acgagctgaa cagcatcgac ctgacacaca
     1201 tcttcatcag ccacaagaag ctggagacaa tcagcagcgc cctgtgcgac cactgggaca
     1261 ccctgagaaa cgccctgtac gagagaagaa tcagcgagct gaccggaaag atcaccaaga
     1321 gcgccaaaga gaaggtccag agaagcctga agcacgagga tatcaacctg caggaaatca
     1381 tcagcgccgc tggaaaagag ctgagcgagg ccttcaagca gaaaaccagc gagatcctgt
     1441 ctcacgccca cgccgccctg gaccagcctc tgcctaccac cctgaagaag caggaagaga
     1501 aagagatcct gaaaagccag ctggacagcc tgctgggact gtaccatctg ctggattggt
     1561 tcgccgtgga cgagagcaac gaggtggacc ctgagttcag cgccagactg accggaatca
     1621 agctggagat ggagcctagc ctgagcttct acaacaaggc tagaaactac gccaccaaga
     1681 aaccctacag cgtggagaag ttcaaactga acttccagat gcctaccctg gccagcggat
     1741 gggacgtgaa caaagagaag aacaacggcg ccatcctgtt cgtgaagaac ggactgtact
     1801 acctgggaat catgcctaag cagaagggaa gatacaaggc tctgagcttc gagcctaccg
     1861 agaaaacaag cgagggattc gacaagatgt actacgatta cttccctgac gccgccaaga
     1921 tgatccctaa gtgcagcacc cagctgaagg ccgtgaccgc ccacttccag acccacacca
     1981 cccctatcct gctgagcaac aacttcatcg agcctctgga gatcaccaaa gagatctacg
     2041 acctgaacaa ccccgagaaa gagcctaaga agttccagac cgcctacgcc aagaaaaccg
     2101 gcgaccagaa gggataccgc gaggctctgt gcaagtggat cgacttcacc agagactttc
     2161 tgagcaagta caccaagacc accagcatcg atctgagcag cctgcgccct agcagccagt
     2221 acaaggacct gggagagtac tacgccgagc tgaaccctct gctgtaccac atcagcttcc
     2281 agagaatcgc cgagaaagag attatggacg ccgtggagac aggcaagctg tacctgttcc
     2341 agatctacaa caaagacttc gccaagggac accacggaaa gcctaacctg cacaccctgt
     2401 actggaccgg actgttcagc cctgagaacc tggctaagac cagcatcaag ctgaacggac
     2461 aggctgagct gttctacaga cctaagagca gaatgaagag aatggcccac agactgggag
     2521 agaagatgct gaacaagaaa ctgaaggacc agaaaacccc tatccccgac acactgtacc
     2581 aggaactgta cgactacgtc aaccacagac tgagccacga cctgtctgac gaggccagag
     2641 ccctgctgcc taacgtgatc acaaaagagg tgtcccacga gatcatcaag gacagacgct
     2701 tcaccagcga caagttcttt ttccacgtgc ccatcaccct gaactaccag gccgccaaca
     2761 gccctagcaa gttcaaccag agagtgaacg cctacctgaa agagcaccct gagacaccta
     2821 tcatcggaat cgacagaggc gagcgcaacc tgatctacat caccgtgatc gacagcaccg
     2881 gaaaaatcct ggagcagaga tccctgaaca ccatccagca gttcgactac cagaagaaac
     2941 tggacaatcg cgagaaagag cgcgtcgccg ccagacaggc ttggagcgtc gtgggaacca
     3001 tcaaggacct gaagcaggga tacctgagcc aggtcatcca cgagatcgtg gacctgatga
     3061 tccactacca ggctgtggtg gtgctggaga atctgaactt cggattcaag agcaagcgca
     3121 ccggaatcgc tgagaaggcc gtgtaccagc agtttgagaa aatgctgatc gacaagctga
     3181 actgcctggt gctgaaggac taccctgccg agaaagtggg aggcgtgctg aacccttacc
     3241 agctgaccga ccagttcacc agcttcgcca agatgggaac ccagagcgga ttcctgttct
     3301 acgtgccagc cccttacacc agcaagatcg accctctgac cggattcgtg gaccctttcg
     3361 tgtggaaaac catcaagaac cacgagagca gaaagcactt cctggagggc ttcgacttcc
     3421 tgcactacga cgtgaaaaca ggcgacttca tcctgcactt caagatgaac agaaacctga
     3481 gcttccagcg cggactgcct ggattcatgc ctgcctggga catcgtgttc gagaaaaacg
     3541 agacacagtt cgacgccaag ggaacccctt ttatcgccgg aaaaagaatc gtgcctgtga
     3601 tcgagaacca cagattcacc ggaagataca gagatctgta ccccgctaac gagctgatcg
     3661 ccctgctgga agagaaggga atcgtgttcc gcgacggaag caacatcctg cctaagctgc
     3721 tggagaacga cgacagccac gctatcgaca caatggtggc cctgatcaga tctgtgctgc
     3781 agatgagaaa cagcaacgcc gccaccggcg aggactacat caacagccct gtgcgcgacc
     3841 tgaacggcgt gtgcttcgac agcagattcc agaaccctga gtggcctatg gacgctgacg
     3901 ccaacggcgc ctaccatatc gccctgaagg gacagctgct gctgaaccat ctgaaagaga
     3961 gcaaagacct gaagctgcag aacggaatca gcaaccagga ctggctggcc tatatccagg
     4021 agctgagaaa caaaagacct gccgctacca agaaggccgg acaggccaag aagaagaagg
     4081 gaagctaccc ttacgacgtg ccagactacg cctacccata cgatgtgcct gattatgctt
     4141 acccatatga cgtgcccgat tacgccggat aaccgcggat ctggttacca ctaaaccagc
     4201 ctcaagaaca cccgaatgga gtctctaagc tacataatac caacttacac tttacaaaat
     4261 gttgtccccc aaaatgtagc cattcgtatc tgctcctaat aaaaagaaag tttcttcaca
     4321 ttctaaaaaa aaaaaaaaaa aaaaaaaaaa aaaccccccc ccccccctgc agggccaagt
     4381 cggcctctag aggtacccaa ttcgccctat agtgagtcgt attacaattc actggccgtc
     4441 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4501 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4561 cagttgcgca gcctgaatgg cgaatggcaa attgtaagcg ttaatatttt gttaaaattc
     4621 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     4681 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     4741 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     4801 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     4861 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     4921 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     4981 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     5041 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5101 atacattcaa atatgtatct gctcatgaga caataaccct gataaatgct tcaataatat
     5161 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5221 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5281 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5341 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5401 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5461 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5521 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5581 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5641 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5701 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5761 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5821 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5881 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5941 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6001 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6061 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6121 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6181 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6241 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6301 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6361 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6421 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6481 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6541 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6601 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6661 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6721 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6781 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6841 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6901 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6961 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7021 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7081 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7141 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     7201 gattacgcca agctcga
//
