LOCUS       sequence-258348-        6997 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Used for in vitro transcription of dual NLS ErCas12a mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6997)
  AUTHORS   Wierson WA, Simone BW, WareJoncas Z, Mann C, Welker JM, Kar B, Emch 
            MJ, Friedberg I, Gendron WAC, Barry MA, Clark KJ, Dobbs DL, McGrail 
            MA, Ekker SC, Essner JJ
  TITLE     Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human 
            Cells.
  JOURNAL   CRISPR J. 2019 Dec;2(6):417-433. doi: 10.1089/crispr.2019.0026. Epub
            2019 Nov 19.
  PUBMED    31742435
REFERENCE   2  (bases 1 to 6997)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1089/crispr.2019.0026"; journalName: "CRISPR J"; date: 
            "2019-12"; volume: "2"; issue: "6"; pages: "417-433"
COMMENT     Sequence Label: pT3TS nErCas12an
FEATURES             Location/Qualifiers
     source          1..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     819..836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    953..974
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(989..994,995..1012,1013..1019)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1027..1043
                     /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     1032..1054
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1051..1067
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1051..1067
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1088..1106
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           1131..1173
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     regulatory      1189..1198
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1207..1227
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5014..5034
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           5059..5184
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(5272..5291)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5273..5291)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5298..5315)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5298..5314)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5307..5329)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(5456..5911)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5593..5614)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5805..5824
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5938..6042
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6043..6111,6112..6903)
                     /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(6261..6280)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
      901 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
      961 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     1021 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     1081 agctcgaaat taaccctcac taaagggaac aaaagctgga gctaagcttg cttgttcttt
     1141 ttgcagaagc tcagaataaa cgctcaactt tggcagatct aactcgacgc caccatggct
     1201 tctccaccta agaagaagag aaaggtgaac aacggaacta ataattttca aaacttcatt
     1261 gggattagtt ctctgcagaa gacccttcgg aatgccctca ttcccactga gacgactcaa
     1321 cagtttatcg taaaaaatgg aattattaag gaggatgagt tgcgggggga gaataggcag
     1381 attttgaaag acatcatgga tgactattac cggggtttta tctccgagac cctgtcctct
     1441 atcgatgata ttgattggac gtctcttttt gagaagatgg agattcagct gaaaaatggt
     1501 gataacaaag ataccctcat taaggagcaa accgagtacc ggaaggcgat ccacaagaag
     1561 ttcgccaacg atgatcgttt taagaatatg ttctcagcca aactcatcag tgacatcctt
     1621 ccagaatttg taattcataa taataactac tctgcgtctg agaaagaaga aaaaactcaa
     1681 gtcatcaagc tcttttcacg gtttgcaacg agctttaagg attactttaa aaaccgcgct
     1741 aattgttttt ctgcggacga catcagctca tccagctgcc acagaatcgt caatgacaat
     1801 gcggagatct tcttctccaa tgctctggta tataggcgca ttgtaaagtc cttgtccaat
     1861 gacgatatta ataagataag tggtgatatg aaggattctc tcaaggaaat gtcattggag
     1921 gagatctaca gctatgagaa atacggtgaa tttattacac aagaaggaat atccttttat
     1981 aatgacatct gtgggaaggt gaattctttc atgaatttgt actgtcaaaa aaacaaggag
     2041 aacaaaaacc tctacaaatt gcaaaaactg cataagcaaa ttctttgtat agcggacact
     2101 agctatgagg tgccgtataa atttgagtca gatgaggagg tctaccaaag cgtaaacggc
     2161 ttcctcgaca acatttccag taagcatatc gtggaacggc tcaggaagat aggggataac
     2221 tataatggct ataaccttga caagatctat atcgtgagta aattctatga aagtgtatct
     2281 caaaagacct atcgagattg ggaaaccata aacacagctc ttgagattca ttacaataat
     2341 attcttcctg gtaacgggaa aagtaaagcc gataaagtga agaaggccgt caaaaacgac
     2401 ctgcagaaga gcataacgga aatcaatgaa ttggtgtcta actacaagct gtgctcagat
     2461 gacaacataa aagctgagac atatatccat gagatcagcc acatactgaa taactttgag
     2521 gcgcaagagc tgaaatataa tcctgagatc caccttgtag agtctgaact caaggcttcc
     2581 gaactgaaaa atgtacttga cgtaatcatg aatgcttttc actggtgtag tgtattcatg
     2641 actgaggaac tggttgataa ggataataat ttttatgcgg aacttgaaga aatatacgat
     2701 gagatttatc ccgttatctc actctataat ttggtccgaa attatgtaac tcaaaaacca
     2761 tactccacaa agaaaatcaa gctcaatttc ggtatcccga ccttggctga cggatggtct
     2821 aaaagcaagg agtactccaa taacgcgata atcttgatgc gagataatct ttactatctc
     2881 ggaattttta atgctaaaaa taagcccgat aaaaagatta ttgaaggaaa cacatctgag
     2941 aacaaaggcg attataaaaa gatgatttat aatttgctcc ctggaccaaa caaaatgatc
     3001 cctaaagttt tcctcagttc caagaccggg gttgagacgt acaagcctag tgcatatatc
     3061 ttggaaggtt ataagcaaaa caagcacatc aaaagttcta aggactttga catcactttt
     3121 tgtcatgatt tgattgacta ttttaaaaac tgtattgcaa ttcacccaga gtggaagaat
     3181 tttggatttg acttctcaga cacgtctacc tatgaagata tatcaggatt ttatcgcgag
     3241 gttgagctcc agggttacaa gattgattgg acttatatca gcgagaagga tattgatctt
     3301 ttgcaggaaa aaggccaact ttatttgttc caaatctaca acaaggactt ttctaagaaa
     3361 tcaactggca acgataacct tcatactatg tacctcaaaa atctcttttc cgaagagaat
     3421 cttaaggata tcgtgctcaa gctgaacggt gaggcagaga tatttttccg aaagagttct
     3481 atcaaaaacc caattatcca caaaaaaggc agcatcctgg ttaacaggac gtacgaggcc
     3541 gaagagaaag atcagttcgg caacatacag atagtgcgga agaatatacc agagaatatc
     3601 taccaagagc tttataagta ttttaatgat aagtcagaca aagagctttc agacgaggcg
     3661 gcgaagttga aaaatgtagt gggacatcac gaagccgcca caaacatcgt gaaggactat
     3721 cggtatacct atgataagta cttccttcac atgccaatca cgatcaattt taaagcgaat
     3781 aagaccgggt tcataaatga ccggattctg cagtacatag caaaggagaa agatcttcat
     3841 gttataggca ttgatcgcgg cgaaagaaac cttatttatg tctccgttat agacacatgt
     3901 gggaacatcg ttgaacaaaa atcctttaat atcgttaatg gatacgacta tcagataaag
     3961 ctcaaacaac aggagggggc gcgccagatt gctcgtaaag aatggaagga aataggaaaa
     4021 ataaaagaaa tcaaggaggg ttacctgagc cttgtaattc atgaaatctc caaaatggtt
     4081 ataaagtaca acgcgattat tgtcatggaa gatttgagct acggttttaa aaaagggagg
     4141 ttcaaggtgg agcgacaggt gtaccaaaag tttgaaacga tgcttattaa taaactcaat
     4201 tacctcgtgt tcaaggatat aagcataaca gaaaatggag ggctccttaa gggataccag
     4261 ctcacataca taccggacaa gcttaaaaac gtgggacacc agtgcgggtg tatattttac
     4321 gttcctgccg cgtatacatc aaagatagac cccaccacag ggttcgtgaa tatcttcaag
     4381 tttaaggact tgacagtcga tgcaaaacgt gagttcatca agaaattcga ttcaatccgg
     4441 tacgattcag aaaagaatct gttctgtttt acgttcgatt ataacaactt tattacgcaa
     4501 aatacagtga tgtcaaagag ctcatggagt gtctacacat acggggttag gataaagcgc
     4561 aggttcgtta acggtcggtt ctcaaacgaa tcagacacga ttgacattac gaaggatatg
     4621 gaaaagactc tggagatgac cgacataaat tggcgagacg gccacgacct ccgacaagat
     4681 atcattgact acgagatcgt ccaacacatt tttgaaatct tccggttgac cgtccagatg
     4741 cgaaacagtc tttctgaatt ggaagaccgg gattacgaca gattgatcag tcctgtattg
     4801 aacgaaaaca acatattcta tgattccgcc aaagctggcg atgctttgcc aaaagacgcc
     4861 gacgcgaatg gagcatattg tatcgccctt aaaggccttt acgaaatcaa acaaataaca
     4921 gagaactgga aagaggatgg gaaatttagc cgagataagc tcaagatcag caacaaagac
     4981 tggtttgact ttattcaaaa caaacggtac ttgccgaaaa agaaacgcaa agtataaccg
     5041 cggatctggt taccactaaa ccagcctcaa gaacacccga atggagtctc taagctacat
     5101 aataccaact tacactttac aaaatgttgt cccccaaaat gtagccattc gtatctgctc
     5161 ctaawaaaaa raaagtttct tcacattcta aaaaaaaaaa aaaaaaaaaa aaaaaaaacc
     5221 cccccccccc cccctgcagg gccaagtcgg cctctagagg tacccaattc gccctatagt
     5281 gagtcgtatt acaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     5341 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     5401 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcaaatt
     5461 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5521 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     5581 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     5641 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     5701 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     5761 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     5821 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     5881 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     5941 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatctgct catgagacaa
     6001 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6061 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6121 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6181 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6241 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6301 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6361 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6421 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6481 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6541 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6601 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6661 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6721 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6781 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6841 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6901 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6961 tttaaaagga tctaggtgaa gatccttttt gataatc
//
