LOCUS       Exported                7025 bp ds-DNA     circular SYN 17-JUN-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7025)
  AUTHORS   Addgene
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Jun 17, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7025
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     promoter        197..408
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      416..425
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             425..490
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             500..520
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             530..550
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             560..580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3545..3565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3842..3862
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3932..4172
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4201..4273
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4288..4428
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4503..4958
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5240..5344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5345..6205
                     /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"
     rep_origin      6376..6964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agctaggtct tgaaaggagt gggaattggc
      181 tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
      241 ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
      301 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
      361 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga ccggtgccac
      421 catggactac aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga
      481 cgatgacaag ggtggatcgc caaagaaaaa gaggaaagtc ggcagtggac ctaaaaagaa
      541 acgaaaggtt gggtcaggtc cgaagaaaaa gcgcaaggtc gaagcgggat ccgcccgcat
      601 cctcgctttc gccatcggaa tctctagtat cggatgggcc ttctctgaaa acgacgaact
      661 gaaagactgc ggcgtgagaa tcttcacaaa ggttgaaaac cctaaaacag gcgagtcttt
      721 agctctgcca cgtaggttgg cccgctccgc ccgaaaaagg ctggctcggc ggaaggctcg
      781 cctcaaccac ttgaagcatt tgatagctaa tgagttcaaa ctgaactacg aagattacca
      841 gtccttcgac gagtcattgg caaaagccta caaaggcagc cttatcagtc cttatgagtt
      901 gagatttcgc gcactcaacg aactgctttc taagcaagac tttgctaggg tcattctgca
      961 catcgcaaaa cggcgaggtt atgacgatat caagaactcc gacgataaag aaaagggagc
     1021 cattctcaag gcgatcaaac agaatgagga aaaattggca aactaccaga gtgtgggcga
     1081 gtatctgtat aaagagtatt tccagaagtt taaggaaaac agcaaggagt ttacaaacgt
     1141 cagaaataaa aaggagtctt acgagagatg catcgcgcag tcattcctca aagatgagct
     1201 gaagctgata tttaagaagc aacgcgaatt tggtttctca ttctctaaga agttcgaaga
     1261 ggaggttctt tccgtggcgt tttacaagag ggcgctcaaa gacttctccc acctggttgg
     1321 taactgtagt ttcttcacgg atgagaagcg agctcccaaa aattctcccc tggctttcat
     1381 gtttgttgcc ctgactcgga tcattaacct gctgaacaac ctgaaaaata ctgaagggat
     1441 cttgtatacg aaggacgacc taaatgcact cctgaatgaa gtgctcaaaa acggaactct
     1501 aacctataaa cagaccaaga aattactggg gctctctgac gactacgagt tcaagggcga
     1561 gaagggtact tattttatcg aattcaaaaa gtataaggag ttcattaaag cattggggga
     1621 acacaacctc agccaggacg atctcaatga aattgccaag gacatcacgc tgattaaaga
     1681 cgagataaaa ctgaaaaagg cactggccaa gtatgacctc aaccagaacc agatcgactc
     1741 tctgtccaag ctggagttca aagaccacct aaacatatcc ttcaaagccc tgaaactggt
     1801 cacccctcta atgctcgaag gaaaaaaata cgacgaggcg tgtaatgaac tgaatcttaa
     1861 ggtggccatc aatgaggata agaaggactt tcttccagcc tttaacgaga catattacaa
     1921 agacgaggtc acaaacccgg ttgtgctgag ggccataaaa gagtatcgga aggttctgaa
     1981 tgccctcctg aagaagtacg gcaaagtgca caaaataaat atcgaattgg ctagggaggt
     2041 ggggaagaac cattctcagc gagcaaagat cgagaaagag cagaatgaga actacaaagc
     2101 caagaaagac gccgaactgg agtgcgaaaa gctggggctt aaaataaaca gtaaaaacat
     2161 cctgaaatta agattgttca aagagcaaaa ggagttttgc gcctactcag gggaaaaaat
     2221 caaaatatca gacctgcagg acgagaaaat gctggagatc gacgccatct atccgtatag
     2281 caggtcattt gacgattcct acatgaacaa agtgcttgtg tttaccaaac agaaccaaga
     2341 aaagctgaac caaaccccct ttgaggcttt cggaaacgac tcagccaagt ggcagaaaat
     2401 cgaagtccta gccaagaatc tgcctacaaa aaaacaaaag aggattcttg ataagaacta
     2461 taaggacaag gaacagaaaa actttaaaga caggaacctg aatgacacga ggtacattgc
     2521 gcgactggtt ctaaactata ccaaagacta cctggatttc ctccctctga gcgacgacga
     2581 gaatactaaa ctgaatgata cccagaaagg ctcaaaggtc cacgttgagg ctaagtccgg
     2641 gatgctgact agcgccctcc gccacacgtg gggcttcagc gccaaagatc ggaataatca
     2701 tcttcatcac gctattgatg cagtaatcat agcctacgct aacaacagca tcgtgaaagc
     2761 cttctccgat ttcaagaaag aacaggagtc taatagcgcc gagttgtacg ccaagaaaat
     2821 ttccgaattg gactataaaa ataagagaaa attcttcgaa cccttctccg ggtttcgcca
     2881 aaaggtctta gataagatcg acgagatttt cgtttccaag cccgaaagaa aaaagccttc
     2941 aggggcactg cacgaagaga cattccgcaa ggaagaggaa ttttaccaat cttacggtgg
     3001 taaagaggga gttctgaagg ctctggagct tgggaagatc cgcaaggtaa acgggaaaat
     3061 cgtgaaaaac ggggacatgt tcagggtgga tatcttcaag cacaaaaaga ccaacaagtt
     3121 ctacgcagta cccatctaca ctatggattt cgctttaaag gttctcccaa ataaggcggt
     3181 ggctcgatcg aagaaaggag agatcaagga ctggatctta atggatgaaa attacgagtt
     3241 ttgcttctcg ctctacaaag atagcctgat tctgatccag acaaaagaca tgcaggaacc
     3301 agaatttgtt tattataacg ccttcacgag cagtacagtg tccctgattg tgagcaagca
     3361 tgataacaag ttcgagactc tgtctaagaa tcagaaaatc cttttcaaga acgccaacga
     3421 gaaggaggtc atcgcaaagt caattggcat ccaaaacctg aaggtgttcg agaaatacat
     3481 agtgtccgca ctcggtgaag taactaaagc cgaatttcga cagcgcgagg attttaagaa
     3541 aagccccaag aagaagagaa aggtggaggc cagcttagca tcgggaggtg gttcgggtgg
     3601 aggaagtggc gggtcagggt cgggtggcac tagtcggggc tttgagagag gactggaacc
     3661 agaaaagatc attggggcaa cagattcctg tggtgattta atgttcctaa tgaaatggaa
     3721 agacacagat gaagctgacc tggttcttgc aaaagaagct aatgtgaaat gtccacaaat
     3781 tgtgatagca ttttatgaag agagactgac atggcatgca tatgctagcg gaggtggttc
     3841 gccaaagaag aaacggaagg tgtgagcatg caataaaaca tctttgtttt cattagatct
     3901 gtgtgttggt tttttgtgtg cggccggtac cgagggccta tttcccatga ttccttcata
     3961 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     4021 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     4081 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     4141 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggagaccacg gcaggtctca
     4201 gttttagtcc ctgaaaaggg actaaaataa agagtttgcg ggactctgcg gggttacaat
     4261 cccctaaaac cgcttttttg cggccgcagg aacccctagt gatggagttg gccactccct
     4321 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     4381 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     4441 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4501 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4561 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4621 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4681 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     4741 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4801 tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt
     4861 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     4921 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     4981 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     5041 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5101 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     5161 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5221 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5281 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5341 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5401 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5461 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5521 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5581 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5641 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5701 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5761 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5821 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5881 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5941 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6001 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6061 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6121 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6181 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6241 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6301 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6361 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6421 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6481 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     6541 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6601 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6661 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6721 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6781 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6841 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6901 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6961 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7021 catgt
//
