LOCUS       Exported                7551 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 7551)
  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..7551
                     /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             3818..4003
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             4367..4387
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4397..4445
                     /note="synthetic polyadenylation signal"
     promoter        4457..4697
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4725..4800
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4814..4954
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5029..5484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5766..5870
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5871..6731
                     /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      6902..7490
                     /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 cactagtcgg acactggtga ccttcaagga
     3841 tgtatttgtg gacttcacca gggaggagtg gaagctgctg gacactgctc agcagatcgt
     3901 gtacagaaat gtgatgctgg agaactataa gaacctggtt tccttgggtt atcagcttac
     3961 taagccagat gtgatcctcc ggttggagaa gggagaagag ccctcgggag gtggttcggg
     4021 aggtggttcg gagggtgtgc aggtgaaaag ggtcctggag aaaagtcctg ggaagctcct
     4081 tgtcaagatg ccttttcaaa cttcgccagg gggcaaggct gaggggggtg gggccaccac
     4141 atccacccag gtcatggtga tcaaacgccc cggcaggaag cgaaaagctg aggccgaccc
     4201 tcaggccatt cccaagaaac ggggccgaaa gccggggagt gtggtggcag ccgctgccgc
     4261 cgaggccaaa aagaaagccg tgaaggagtc ttctatccga tctgtgcagg agacagtact
     4321 ccccatcaag aagcgcaaga cccgggaggc tagcggaggt ggttcgccaa agaagaaacg
     4381 gaaggtggga tcctgaaata aaagatcttt gttttcatta gatctgtgtg ttggtttttt
     4441 gtgtgcggcc ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
     4501 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     4561 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     4621 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     4681 tatcttgtgg aaaggacgaa acaccgagga gggggcggga cagggtttta gtactctgga
     4741 aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga
     4801 tttttgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     4861 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     4921 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     4981 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     5041 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5101 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5161 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5221 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     5281 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     5341 aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc
     5401 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5461 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     5521 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5581 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5641 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     5701 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     5761 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     5821 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     5881 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5941 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6001 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6061 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6121 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6181 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6241 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     6301 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     6361 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     6421 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     6481 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     6541 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     6601 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     6661 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     6721 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     6781 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6841 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6901 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6961 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7021 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     7081 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7141 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7201 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7261 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     7321 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     7381 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     7441 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     7501 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
