LOCUS       Exported                7213 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 7213)
  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..7213
                     /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             524..544
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3725..3772
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4028..4048
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4058..4106
                     /note="synthetic polyadenylation signal"
     promoter        4118..4358
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4387..4462
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4476..4616
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4691..5146
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5428..5532
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5533..6393
                     /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      6564..7152
                     /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 gacatagatt acaaggatga
      481 cgatgacaag ggtggatcgc caaagaaaaa gaggaaagtc gccccaaaga agaagcggaa
      541 ggtcggtatc cacggagtcc cagcagccaa gcggaactac atcctgggcc tggccatcgg
      601 catcaccagc gtgggctacg gcatcatcga ctacgagaca cgggacgtga tcgatgccgg
      661 cgtgcggctg ttcaaagagg ccaacgtgga aaacaacgag ggcaggcgga gcaagagagg
      721 cgccagaagg ctgaagcggc ggaggcggca tagaatccag agagtgaaga agctgctgtt
      781 cgactacaac ctgctgaccg accacagcga gctgagcggc atcaacccct acgaggccag
      841 agtgaagggc ctgagccaga agctgagcga ggaagagttc tctgccgccc tgctgcacct
      901 ggccaagaga agaggcgtgc acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct
      961 gtccaccaaa gagcagatca gccggaacag caaggccctg gaagagaaat acgtggccga
     1021 actgcagctg gaacggctga agaaagacgg cgaagtgcgg ggcagcatca acagattcaa
     1081 gaccagcgac tacgtgaaag aagccaaaca gctgctgaag gtgcagaagg cctaccacca
     1141 gctggaccag agcttcatcg acacctacat cgacctgctg gaaacccggc ggacctacta
     1201 tgagggacct ggcgagggca gccccttcgg ctggaaggac atcaaagaat ggtacgagat
     1261 gctgatgggc cactgcacct acttccccga ggaactgcgg agcgtgaagt acgcctacaa
     1321 cgccgacctg tacaacgccc tgaacgacct gaacaatctc gtgatcacca gggacgagaa
     1381 cgagaagctg gaatattacg agaagttcca gatcatcgag aacgtgttca agcagaagaa
     1441 gaagcccacc ctgaagcaga tcgccaaaga aatcctcgtg aacgaagagg atattaaggg
     1501 ctacagagtg accagcaccg gcaagcccga gttcaccaac ctgaaggtgt accacgacat
     1561 caaggacatt accgcccgga aagagattat tgagaacgcc gagctgctgg atcagattgc
     1621 caagatcctg accatctacc agagcagcga ggacatccag gaagaactga ccaatctgaa
     1681 ctccgagctg acccaggaag agatcgagca gatctctaat ctgaagggct ataccggcac
     1741 ccacaacctg agcctgaagg ccatcaacct gatcctggac gagctgtggc acaccaacga
     1801 caaccagatc gctatcttca accggctgaa gctggtgccc aagaaggtgg acctgtccca
     1861 gcagaaagag atccccacca ccctggtgga cgacttcatc ctgagccccg tcgtgaagag
     1921 aagcttcatc cagagcatca aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa
     1981 cgacatcatt atcgagctgg cccgcgagaa gaactccaag gacgcccaga aaatgatcaa
     2041 cgagatgcag aagcggaacc ggcagaccaa cgagcggatc gaggaaatca tccggaccac
     2101 cggcaaagag aacgccaagt acctgatcga gaagatcaag ctgcacgaca tgcaggaagg
     2161 caagtgcctg tacagcctgg aagccatccc tctggaagat ctgctgaaca accccttcaa
     2221 ctatgaggtg gaccacatca tccccagaag cgtgtccttc gacaacagct tcaacaacaa
     2281 ggtgctcgtg aagcaggaag aagccagcaa gaagggcaac cggaccccat tccagtacct
     2341 gagcagcagc gacagcaaga tcagctacga aaccttcaag aagcacatcc tgaatctggc
     2401 caagggcaag ggcagaatca gcaagaccaa gaaagagtat ctgctggaag aacgggacat
     2461 caacaggttc tccgtgcaga aagacttcat caaccggaac ctggtggata ccagatacgc
     2521 caccagaggc ctgatgaacc tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa
     2581 agtgaagtcc atcaatggcg gcttcaccag ctttctgcgg cggaagtgga agtttaagaa
     2641 agagcggaac aaggggtaca agcaccacgc cgaggacgcc ctgatcattg ccaacgccga
     2701 tttcatcttc aaagagtgga agaaactgga caaggccaaa aaagtgatgg aaaaccagat
     2761 gttcgaggaa aagcaggccg agagcatgcc cgagatcgaa accgagcagg agtacaaaga
     2821 gatcttcatc accccccacc agatcaagca cattaaggac ttcaaggact acaagtacag
     2881 ccaccgggtg gacaagaagc ctaatagaga gctgattaac gacaccctgt actccacccg
     2941 gaaggacgac aagggcaaca ccctgatcgt gaacaatctg aacggcctgt acgacaagga
     3001 caatgacaag ctgaaaaagc tgatcaacaa gagccccgaa aagctgctga tgtaccacca
     3061 cgacccccag acctaccaga aactgaagct gattatggaa cagtacggcg acgagaagaa
     3121 tcccctgtac aagtactacg aggaaaccgg gaactacctg accaagtact ccaaaaagga
     3181 caacggcccc gtgatcaaga agattaagta ttacggcaac aaactgaacg cccatctgga
     3241 catcaccgac gactacccca acagcagaaa caaggtcgtg aagctgtccc tgaagcccta
     3301 cagattcgac gtgtacctgg acaatggcgt gtacaagttc gtgaccgtga agaatctgga
     3361 tgtgatcaaa aaagaaaact actacgaagt gaatagcaag tgctatgagg aagctaagaa
     3421 gctgaagaag atcagcaacc aggccgagtt tatcgcctcc ttctacaaca acgatctgat
     3481 caagatcaac ggcgagctgt atagagtgat cggcgtgaac aacgacctgc tgaaccggat
     3541 cgaagtgaac atgatcgaca tcacctaccg cgagtacctg gaaaacatga acgacaagag
     3601 gccccccagg atcattaaga caatcgcctc caagacccag agcattaaga agtacagcac
     3661 agacattctg ggcaacctgt atgaagtgaa atctaagaag caccctcaga tcatcaaaaa
     3721 gggcaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccgg
     3781 tggaggaagt ggcgggtcag ggtcgggtgg cactagtgca gtagacccag gcctgccttc
     3841 tgtgaagcaa gagccacctg acccagagga ggacaaggag gagaacaagg atgattctgc
     3901 ctccaaattg gccccagagg aagaggcagg aggggctggc acacccgtga tcacggagat
     3961 tttcagcctg ggtggaaccc gcttccgaga tacagcagtc tggttgccag ctagcggagg
     4021 tggttcgcca aagaagaaac ggaaggtggg atcctgaaat aaaagatctt tgttttcatt
     4081 agatctgtgt gttggttttt tgtgtgcggc cggtaccgag ggcctatttc ccatgattcc
     4141 ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt
     4201 aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt
     4261 tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta
     4321 tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccggag accacggcag
     4381 gtctcagttt tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta
     4441 tctcgtcaac ttgttggcga gatttttgcg gccgcaggaa cccctagtga tggagttggc
     4501 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4561 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4621 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4681 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4741 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4801 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4861 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4921 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4981 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     5041 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5101 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5161 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5221 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5281 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5341 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5401 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5461 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5521 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5581 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5641 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5701 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5761 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5821 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5881 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5941 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6001 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6061 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6121 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6181 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6241 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6301 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6361 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6421 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6481 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6541 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6601 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6661 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6721 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6781 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6841 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6901 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6961 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7021 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7081 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7141 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7201 tttgctcaca tgt
//
