LOCUS       Exported                7189 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 7189)
  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..7189
                     /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             4004..4024
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4034..4082
                     /note="synthetic polyadenylation signal"
     promoter        4094..4334
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4363..4438
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4452..4592
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4667..5122
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5404..5508
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5509..6369
                     /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      6540..7128
                     /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 cactagtgat gtctactact tcagtccaag
     3841 tggtaagaag ttcagaagca agcctcagtt ggcaaggtac ctgggaaata ctgttgatct
     3901 cagcagtttt gacttcagaa ctggaaagat gatgcctagt aaattacaga agaacaaaca
     3961 gagactgcga aacgatcctc tcaatgctag cggaggtggt tcgccaaaga agaaacggaa
     4021 ggtgggatcc tgaaataaaa gatctttgtt ttcattagat ctgtgtgttg gttttttgtg
     4081 tgcggccggt accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     4141 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     4201 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     4261 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     4321 cttgtggaaa ggacgaaaca ccggagacca cggcaggtct cagttttagt actctggaaa
     4381 cagaatctac taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt
     4441 tttgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4501 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4561 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     4621 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4681 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4741 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4801 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4861 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4921 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4981 actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg
     5041 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5101 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     5161 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5221 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5281 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     5341 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5401 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5461 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5521 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5581 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5641 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5701 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5761 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5821 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5881 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5941 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     6001 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     6061 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     6121 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     6181 gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt
     6241 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6301 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6361 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6421 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6481 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6541 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6601 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6661 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     6721 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6781 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6841 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6901 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6961 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7021 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7081 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7141 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
