LOCUS       Exported                7121 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 7121)
  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..7121
                     /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             3938..3958
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        4028..4268
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4297..4369
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4384..4524
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4599..5054
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5336..5440
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5441..6301
                     /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      6472..7060
                     /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 tagtaaatca gagggaggca agcgcaaagc
     3661 tgattctgat tctgaagata agggagagga gagcaaacca aagaagaaga aagaagagtc
     3721 agaaaagcca cgaggctttg ctcgaggttt ggagccggag cggattattg gagctacaga
     3781 ctccagtgga gagctcatgt tcctgatgaa atggaaaaac tctgatgagg ctgacctggt
     3841 ccctgccaag gaagccaatg tcaagtgccc acaggttgtc atatccttct atgaggaaag
     3901 gctgacgtgg cattcctacg ctagcggagg tggttcgcca aagaagaaac ggaaggtgtg
     3961 agcatgcaat aaaacatctt tgttttcatt agatctgtgt gttggttttt tgtgtgcggc
     4021 cggtaccgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     4081 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     4141 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     4201 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     4261 gaaaggacga aacaccggag accacggcag gtctcagttt tagtccctga aaagggacta
     4321 aaataaagag tttgcgggac tctgcggggt tacaatcccc taaaaccgct tttttgcggc
     4381 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4441 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4501 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4561 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4621 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4681 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4741 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4801 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4861 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4921 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc
     4981 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5041 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5101 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5161 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5221 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5281 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5341 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5401 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5461 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5521 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5581 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5641 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5701 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5761 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5821 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5881 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5941 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6001 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6061 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6121 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     6181 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6241 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6301 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6361 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6421 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6481 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6541 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6601 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6661 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6721 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6781 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6841 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6901 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6961 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7021 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7081 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
