LOCUS       Exported                7082 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 7082)
  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..7082
                     /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             3899..3919
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3989..4229
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4258..4330
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4345..4485
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4560..5015
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5297..5401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5402..6262
                     /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      6433..7021
                     /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 tagtatgggt ggagaggatg atgaactcaa
     3661 gggcttactg gggcttccag aggaggaaac tgagcttgat aacctgacag agttcaacac
     3721 tgcccacaac aagcggattt ctacccttac cattgaggag ggaaatctgg acattcaaag
     3781 accaaagagg aagaggaaga actcacgggt gacattcagt gaggatgatg agatcatcaa
     3841 cccagaggat gtggatccct cagttggtcg attcaggaac gctagcggag gtggttcgcc
     3901 aaagaagaaa cggaaggtgt gagcatgcaa taaaacatct ttgttttcat tagatctgtg
     3961 tgttggtttt ttgtgtgcgg ccggtaccga gggcctattt cccatgattc cttcatattt
     4021 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     4081 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     4141 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     4201 cttggcttta tatatcttgt ggaaaggacg aaacaccgga gaccacggca ggtctcagtt
     4261 ttagtccctg aaaagggact aaaataaaga gtttgcggga ctctgcgggg ttacaatccc
     4321 ctaaaaccgc ttttttgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     4381 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4441 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4501 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4561 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4621 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4681 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4741 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4801 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4861 actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata
     4921 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4981 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5041 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     5101 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5161 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     5221 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     5281 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5341 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5401 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5461 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5521 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5581 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5641 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5701 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5761 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5821 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5881 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5941 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6001 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6061 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     6121 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6181 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6241 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6301 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6361 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6421 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6481 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6541 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6601 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6661 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6721 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6781 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6841 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6901 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6961 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7021 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7081 gt
//
