LOCUS       pCAG-eSpCas9-2A-GFP-sgRNA-DNA-PKcs       10245 bp    DNA     circular SYN 29-APR-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
SOURCE      synthetic DNA construct.
  ORGANISM  recombinant plasmid
FEATURES             Location/Qualifiers
     source          1..10245
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
                     /vntifkey="98"
     promoter        1..241
                     /vntifkey="30"
                     /label=U6\promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        270..345
                     /vntifkey="53"
                     /label=gRNA\scaffold
                     /note="guide RNA scaffold for the CRISPR/Cas9 system"
     enhancer        525..828
                     /vntifkey="9"
                     /label=CMV\enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        830..1107
                     /vntifkey="30"
                     /label=chicken\beta-actin\promoter
     CDS             2210..2275
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /vntifkey="4"
                     /label=3xFLAG
     CDS             2282..2302
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /vntifkey="4"
                     /label=SV40\NLS
     CDS             2327..6427
                     /codon_start=1
                     /vntifkey="4"
                     /label=eCas9
     CDS             6428..6475
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /vntifkey="4"
                     /label=nucleoplasmin\NLS
     CDS             6491..6544
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /vntifkey="4"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides."
     CDS             6545..7258
                     /codon_start=1
                     /product="enhanced GFP"
                     /vntifkey="4"
                     /label=EGFP
                     /note="mammalian codon-optimized"
     polyA_signal    7292..7499
                     /vntifkey="25"
                     /label=bGH\poly(A)\signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7508..7648
                     /vntifkey="34"
                     /label=AAV2\ITR
                     /note="inverted terminal repeat of adeno-associated virus serotype 2"
     rep_origin      7723..8178
                     /direction=RIGHT
                     /vntifkey="33"
                     /label=f1\ori
                     /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis"
     promoter        8460..8564
                     /gene="bla"
                     /vntifkey="30"
                     /label=AmpR\promoter
     CDS             8565..9425
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /vntifkey="4"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and related antibiotics"
     rep_origin      9596..10184
                     /direction=RIGHT
                     /vntifkey="33"
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"
     primer_bind     10244..23
                     /vntifkey="28"
                     /label=U6-F
     misc_feature    250..269
                     /vntifkey="21"
                     /label=sgRNA-hDNA-PKcs
BASE COUNT     2505 a      2777 c      2939 g      2024 t 
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 
      241 cgaaacaccg gtacccaccc agcaccgcgg ttttagagct agaaatagca agttaaaata 
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 
      421 acaaatggct ctagtaacgg ccgccagtgt gctggaattc aggctagtta ttaatagtaa 
      481 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 
      541 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 
      601 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta 
      661 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 
      721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 
      781 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc 
      841 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta 
      901 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca 
      961 ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc 
     1021 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc 
     1081 tataaaaagc gaagcgcgcg gcgggcgggg agtcgctgcg acgctgcctt cgccccgtgc 
     1141 cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac 
     1201 aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac 
     1261 ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt 
     1321 gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg 
     1381 gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc 
     1441 gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg 
     1501 aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg 
     1561 cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag cacggcccgg 
     1621 cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg ggcggggggt 
     1681 ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggag ggctcggggg 
     1741 aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt 
     1801 gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa atctgtgcgg 
     1861 agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg 
     1921 gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc 
     1981 ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg 
     2041 gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg 
     2101 ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc 
     2161 atcattttgg caaagaattc ctgaattctg cagatccggt gccaccatgg actataagga 
     2221 ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc 
     2281 cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag 
     2341 catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa 
     2401 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa 
     2461 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag 
     2521 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt 
     2581 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct 
     2641 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt 
     2701 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac 
     2761 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg 
     2821 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat 
     2881 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt 
     2941 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat 
     3001 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct 
     3061 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct 
     3121 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta 
     3181 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct 
     3241 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga 
     3301 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa 
     3361 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg 
     3421 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac 
     3481 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga 
     3541 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca 
     3601 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt 
     3661 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac 
     3721 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc 
     3781 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa 
     3841 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa 
     3901 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa 
     3961 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga 
     4021 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg 
     4081 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt 
     4141 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt 
     4201 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa 
     4261 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct 
     4321 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga 
     4381 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga 
     4441 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa 
     4501 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga 
     4561 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga 
     4621 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga 
     4681 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct 
     4741 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca 
     4801 ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt 
     4861 tctggccgac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa 
     4921 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct 
     4981 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg 
     5041 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca 
     5101 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa 
     5161 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt 
     5221 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga 
     5281 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtaccctg cgctggaaag 
     5341 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga 
     5401 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt 
     5461 caagaccgag attaccctgg ccaacggcga gatccggaag gcgcctctga tcgagacaaa 
     5521 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt 
     5581 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag 
     5641 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg 
     5701 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt 
     5761 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat 
     5821 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg 
     5881 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct 
     5941 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact 
     6001 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa 
     6061 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct 
     6121 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa 
     6181 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc 
     6241 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta 
     6301 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac 
     6361 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg 
     6421 aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt 
     6481 cggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg 
     6541 cccagtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga 
     6601 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta 
     6661 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac 
     6721 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa 
     6781 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt 
     6841 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct 
     6901 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca 
     6961 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa 
     7021 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc 
     7081 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca 
     7141 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt 
     7201 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga 
     7261 attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 
     7321 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 
     7381 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 
     7441 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag 
     7501 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 
     7561 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 
     7621 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat 
     7681 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 
     7741 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct 
     7801 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 
     7861 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 
     7921 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 
     7981 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 
     8041 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 
     8101 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 
     8161 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 
     8221 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 
     8281 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 
     8341 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 
     8401 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 
     8461 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 
     8521 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 
     8581 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 
     8641 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 
     8701 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 
     8761 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 
     8821 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 
     8881 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 
     8941 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 
     9001 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 
     9061 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 
     9121 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 
     9181 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 
     9241 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag 
     9301 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 
     9361 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 
     9421 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 
     9481 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 
     9541 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 
     9601 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 
     9661 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 
     9721 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 
     9781 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 
     9841 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 
     9901 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 
     9961 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 
    10021 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 
    10081 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 
    10141 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 
    10201 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt 
//
