LOCUS       sequence-95666-e        8749 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Eef1a (EF1alpha) promoter driving nls::Cas9::nls.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8749)
  AUTHORS   Stolfi A, Gandhi S, Salek F, Christiaen L
  TITLE     Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9.
  JOURNAL   Development. 2014 Nov;141(21):4115-20. doi: 10.1242/dev.114488.
  PUBMED    25336740
REFERENCE   2  (bases 1 to 8749)
  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"; doi: "10.1242/dev"; 
            journalName: "Development"; date: "2014-11"; volume: "141"; issue: 
            "21"; pages: "4115-20"
COMMENT     Sequence Label: Eef1a-1955/-1>nls::Cas9::nls
FEATURES             Location/Qualifiers
     source          1..8749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1994..2014
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2015..4528,4529..4531,4532..6115)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6125..6145
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6475..6609
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6512..6531)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6566..6585
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6645..6664)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6646..6664)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6751..6768)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6922..7510)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7002..7021)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7681..8472,8473..8541))
                     /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     8304..8323
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8542..8646)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8714..8732
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggcgcgccgt gacgggaaaa cgatagtcgt tataacacga gtattcatac acctcgtgcg
       61 agcttacgag ctaccgtata tgttactttg tgggcgaata aaggttttat aaatataaca
      121 ttggttttat aaataaaaca acgccatttt aaagtcggtt acataattct gtaactagtt
      181 caaattgaac ggtaaacgta aataaaaacc ttgaccgtct tacccaatta tataaaaaca
      241 ctttgaacgc tttttaagat ggaagggtat ggccatgcct agataattct gtggaccatc
      301 tcaccccaac ctattacaga acggtcgtaa taatgaaaat ggataccatt ttaggcatat
      361 agactggttc ctctactttc tagaaacgta agcagtatac acagaaaaaa tgaagtgtgt
      421 ttctgtgcaa attaaaccgt tctaaattca tagccgactg aatttctaat taggtgctga
      481 atggctgacc tagatttatt gttaagttta gcaccaaatc tgagccagcg ataagcagtc
      541 taattaaatt ggctgctggc gataaaatag gtcatcctga aaaatcgttt gcgcctttat
      601 ttaaaatata gtagagtggg gaaagacggg acatcttatc gttctatttt ctcgtcccat
      661 ttcgtagtaa acaaagaaca ttcaaaaaat ataaaaccat aacttcaaaa cttcaataga
      721 ccgttgtcaa ctgtttaaaa cacaataaga gaatttggat attatgtgct aaaggtgtcc
      781 catctccccc caccctacta tatctgttta tagttctgtg gggtaagatg agataccgtt
      841 aacacctaaa catttttact ttaaacaatc aaccacgttt tttatagtcg taatggacat
      901 gtggttacat aattctgaaa atattttttg cccccgacca aaagacgcga agagtaaaaa
      961 catgtctcag cttatattcc ccacataaat atatttttgt actgtttggt gaatttataa
     1021 acttatatta ccatgcatat acgttatgtt actggtattt tctcagtagg caaattcatt
     1081 tgtccacgtt ttataggttt tcaatattta tgatttttaa aatgctaaaa atgtgggagg
     1141 ggggttgaaa gtacaataca aacacacaaa acaactcaaa ctaaagattt atagttatgc
     1201 taattcacct acacaatata acaagatgtg taatgcaacc atgtgtttat gatgagcgct
     1261 aacatatttt gtaaccactc aaattccccg ccacacgagg ataatgaata ggtgactctg
     1321 tagtctgtac atcttagact gaaataaaga ttataaatct acgaaataaa ataatttctg
     1381 ctcactgatt atacttctgt tttatagatt agaaaccgtt tctaataaat gacctaattc
     1441 gctatacaca cacgctgtgc gcgagataat cattctcgca ccccgtttat tgtgttaaaa
     1501 ttgccgccta gattcacaaa gcgtgacggc tagagccagc aacgtgtcgc cttcaattac
     1561 gcaacatccg ggttgcgcaa ttctggatat aaaagaacta acaaagatga cgtagctacc
     1621 tttttcagtt cagacttacg aaagactcac gtgtcggcgg tctacttgtc cttttcgagc
     1681 tgtggcaatt tggtgagtgg ttctatcttt tatctgagta catctctaag gaattatagt
     1741 ttgattagtt aaatttttat tgttaggaaa gatgaaatca ttaggtttta cttagtttaa
     1801 gtatgttagt actggttagg cgtttgaatt actgaaaaac tcagttcgtt aactgtagta
     1861 gttctggtag cttagcaagt ataccctgta tacgcctttt ggctttttaa caataactta
     1921 aacttatttt acagcaaatt tctgtgcatt cggttaaccc caaccttcca aagcggccgc
     1981 aaccatggct agccccaaaa agaagaggaa agtggacaag aagtattcta tcggactgga
     2041 catcgggact aatagcgtcg ggtgggccgt gatcactgac gagtacaagg tgccctctaa
     2101 gaagttcaag gtgctcggga acaccgaccg gcattccatc aagaaaaatc tgatcggagc
     2161 tctcctcttt gattcagggg agaccgctga agcaacccgc ctcaagcgga ctgctagacg
     2221 gcggtacacc aggaggaaga accggatttg ttaccttcaa gagatattct ccaacgaaat
     2281 ggcaaaggtc gacgacagct tcttccatag gctggaagaa tcattcctcg tggaagagga
     2341 taagaagcat gaacggcatc ccatcttcgg taatatcgtc gacgaggtgg cctatcacga
     2401 gaaataccca accatctacc atcttcgcaa aaagctggtg gactcaaccg acaaggcaga
     2461 cctccggctt atctacctgg ccctggccca catgatcaag ttcagaggcc acttcctgat
     2521 cgagggcgac ctcaatcctg acaatagcga tgtggataaa ctgttcatcc agctggtgca
     2581 gacttacaac cagctctttg aagagaaccc catcaatgca agcggagtcg atgccaaggc
     2641 cattctgtca gcccggctgt caaagagccg cagacttgag aatcttatcg ctcagctgcc
     2701 gggtgaaaag aaaaatggac tgttcgggaa cctgattgct ctttcacttg ggctgactcc
     2761 caatttcaag tctaatttcg acctggcaga ggatgccaag ctgcaactgt ccaaggacac
     2821 ctatgatgac gatctcgaca acctcctggc ccagatcggt gaccaatacg ccgacctttt
     2881 ccttgctgct aagaatcttt ctgacgccat cctgctgtct gacattctcc gcgtgaacac
     2941 tgaaatcacc aaggcccctc tttcagcttc aatgattaag cggtatgatg agcaccacca
     3001 ggacctgacc ctgcttaagg cactcgtccg gcagcagctt ccggagaagt acaaggaaat
     3061 cttctttgac cagtcaaaga atggatacgc cggctacatc gacggaggtg cctcccaaga
     3121 ggaattttat aagtttatca aacctatcct tgagaagatg gacggcaccg aagagctcct
     3181 cgtgaaactg aatcgggagg atctgctgcg gaagcagcgc actttcgaca atgggagcat
     3241 tccccaccag atccatcttg gggagcttca cgccatcctt cggcgccaag aggacttcta
     3301 cccctttctt aaggacaaca gggagaagat tgagaaaatt ctcactttcc gcatccccta
     3361 ctacgtggga cccctcgcca gaggaaatag ccggtttgct tggatgacca gaaagtcaga
     3421 agaaactatc actccctgga acttcgaaga ggtggtggac aagggagcca gcgctcagtc
     3481 attcatcgaa cggatgacta acttcgataa gaacctcccc aatgagaagg tcctgccgaa
     3541 acattccctg ctctacgagt actttaccgt gtacaacgag ctgaccaagg tgaaatatgt
     3601 caccgaaggg atgaggaagc ccgcattcct gtcaggcgaa caaaagaagg caattgtgga
     3661 ccttctgttc aagaccaata gaaaggtgac cgtgaagcag ctgaaggagg actatttcaa
     3721 gaaaattgaa tgcttcgact ctgtggagat tagcggggtc gaagatcggt tcaacgcaag
     3781 cctgggtacc taccatgatc tgcttaagat catcaaggac aaggattttc tggacaatga
     3841 ggagaacgag gacatccttg aggacattgt cctgactctc actctgttcg aggaccggga
     3901 aatgatcgag gagaggctta agacctacgc ccatctgttc gacgataaag tgatgaagca
     3961 acttaaacgg agaagatata ccggatgggg acgccttagc cgcaaactca tcaacggaat
     4021 ccgggacaaa cagagcggaa agaccattct tgatttcctt aagagcgacg gattcgctaa
     4081 tcgcaacttc atgcaactta tccatgatga ttccctgacc tttaaggagg acatccagaa
     4141 ggcccaagtg tctggacaag gtgactcact gcacgagcat atcgcaaatc tggctggttc
     4201 acccgctatt aagaagggta ttctccagac cgtgaaagtc gtggacgagc tggtcaaggt
     4261 gatgggtcgc cataaaccag agaacattgt catcgagatg gccagggaaa accagactac
     4321 ccagaaggga cagaagaaca gcagggagcg gatgaaaaga attgaggaag ggattaagga
     4381 gctcgggtca cagatcctta aagagcaccc ggtggaaaac acccagcttc agaatgagaa
     4441 gctctatctg tactaccttc aaaatggacg cgatatgtat gtggaccaag agcttgatat
     4501 caacaggctc tcagactacg acgtggacgc catcgtccct cagagcttcc tcaaagacga
     4561 ctcaattgac aataaggtgc tgactcgctc agacaagaac cggggaaagt cagataacgt
     4621 gccctcagag gaagtcgtga aaaagatgaa gaactattgg cgccagcttc tgaacgcaaa
     4681 gctgatcact cagcggaagt tcgacaatct cactaaggct gagaggggcg gactgagcga
     4741 actggacaaa gcaggattca ttaaacggca acttgtggag actcggcaga ttactaaaca
     4801 tgtcgcccaa atccttgact cacgcatgaa taccaagtac gacgaaaacg acaaacttat
     4861 ccgcgaggtg aaggtgatta ccctgaagtc caagctggtc agcgatttca gaaaggactt
     4921 tcaattctac aaagtgcggg agatcaataa ctatcatcat gctcatgacg catatctgaa
     4981 tgccgtggtg ggaaccgccc tgatcaagaa gtacccaaag ctggaaagcg agttcgtgta
     5041 cggagactac aaggtctacg acgtgcgcaa gatgattgcc aaatctgagc aggagatcgg
     5101 aaaggccacc gcaaagtact tcttctacag caacatcatg aatttcttca agaccgaaat
     5161 cacccttgca aacggtgaga tccggaagag gccgctcatc gagactaatg gggagactgg
     5221 cgaaatcgtg tgggacaagg gcagagattt cgctaccgtg cgcaaagtgc tttctatgcc
     5281 tcaagtgaac atcgtgaaga aaaccgaggt gcaaaccgga ggcttttcta aggaatcaat
     5341 cctccccaag cgcaactccg acaagctcat tgcaaggaag aaggattggg accctaagaa
     5401 gtacggcgga ttcgattcac caactgtggc ttattctgtc ctggtcgtgg ctaaggtgga
     5461 aaaaggaaag tctaagaagc tcaagagcgt gaaggaactg ctgggtatca ccattatgga
     5521 gcgcagctcc ttcgagaaga acccaattga ctttctcgaa gccaaaggtt acaaggaagt
     5581 caagaaggac cttatcatca agctcccaaa gtatagcctg ttcgaactgg agaatgggcg
     5641 gaagcggatg ctcgcctccg ctggcgaact tcagaagggt aatgagctgg ctctcccctc
     5701 caagtacgtg aatttcctct accttgcaag ccattacgag aagctgaagg ggagccccga
     5761 ggacaacgag caaaagcaac tgtttgtgga gcagcataag cattatctgg acgagatcat
     5821 tgagcagatt tccgagtttt ctaaacgcgt cattctcgct gatgccaacc tcgataaagt
     5881 ccttagcgca tacaataagc acagagacaa accaattcgg gagcaggctg agaatatcat
     5941 ccacctgttc accctcacca atcttggtgc ccctgccgca ttcaagtact tcgacaccac
     6001 catcgaccgg aaacgctata cctccaccaa agaagtgctg gacgccaccc tcatccacca
     6061 gagcatcacc ggactttacg aaactcggat tgacctctca cagctcggag gggatgaggg
     6121 agctcccaag aaaaagcgca aggtaggtta atgagaattc cagctgagcg ccggtcgcta
     6181 ccattaccag ttggtctggt gtcaaaaata ataataaccg ggcaggccat gtctgcccgt
     6241 atttcgcgta aggaaatcca ttatgtacta tttaaaaaac acaaactttt ggatgttcgg
     6301 tttattcttt ttcttttact tttttatcat gggagcctac ttcccgtttt tcccgatttg
     6361 gctacatgac atcaaccata tcagcaaaag tgatacgggt attatttttg ccgctatttc
     6421 tctgttctcg ctattattcc aaccgctgtt tggtctgctt tctgacaaac tcggaacttg
     6481 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     6541 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     6601 gtctggatcg acaaagtcaa agcggccatc agatctgccg gtctccctat agtgagtcgt
     6661 attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg cggggagagg
     6721 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6781 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6841 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6901 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6961 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     7021 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     7081 cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
     7141 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     7201 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     7261 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7321 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     7381 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7441 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7501 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7561 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7621 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7681 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7741 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7801 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7861 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7921 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7981 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     8041 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     8101 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     8161 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     8221 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     8281 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     8341 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     8401 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     8461 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8521 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     8581 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     8641 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     8701 attaacctat aaaaataggc gtatcacgag gcccttacgt attaattaa
//
