LOCUS       Exported                7367 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 7367)
  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..7367
                     /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             3635..3820
                     /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             4184..4204
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        4274..4514
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4543..4615
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4630..4770
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4845..5300
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5582..5686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5687..6547
                     /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      6718..7306
                     /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 tagtcggaca ctggtgacct tcaaggatgt
     3661 atttgtggac ttcaccaggg aggagtggaa gctgctggac actgctcagc agatcgtgta
     3721 cagaaatgtg atgctggaga actataagaa cctggtttcc ttgggttatc agcttactaa
     3781 gccagatgtg atcctccggt tggagaaggg agaagagccc tcgggaggtg gttcgggagg
     3841 tggttcggag ggtgtgcagg tgaaaagggt cctggagaaa agtcctggga agctccttgt
     3901 caagatgcct tttcaaactt cgccaggggg caaggctgag gggggtgggg ccaccacatc
     3961 cacccaggtc atggtgatca aacgccccgg caggaagcga aaagctgagg ccgaccctca
     4021 ggccattccc aagaaacggg gccgaaagcc ggggagtgtg gtggcagccg ctgccgccga
     4081 ggccaaaaag aaagccgtga aggagtcttc tatccgatct gtgcaggaga cagtactccc
     4141 catcaagaag cgcaagaccc gggaggctag cggaggtggt tcgccaaaga agaaacggaa
     4201 ggtgtgagca tgcaataaaa catctttgtt ttcattagat ctgtgtgttg gttttttgtg
     4261 tgcggccggt accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     4321 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     4381 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     4441 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     4501 cttgtggaaa ggacgaaaca ccggagacca cggcaggtct cagttttagt ccctgaaaag
     4561 ggactaaaat aaagagtttg cgggactctg cggggttaca atcccctaaa accgcttttt
     4621 tgcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     4681 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     4741 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     4801 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4861 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4921 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4981 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5041 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5101 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5161 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     5221 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5281 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     5341 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5401 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5461 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5521 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5581 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5641 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5701 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5761 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5821 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5881 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5941 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6001 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6061 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6121 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6181 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6241 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6301 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6361 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     6421 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6481 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6541 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6601 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6661 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6721 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6781 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6841 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     6901 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6961 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7021 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7081 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7141 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7201 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7261 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7321 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
