LOCUS       Exported                7028 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 7028)
  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..7028
                     /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             3845..3865
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        3935..4175
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4204..4276
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   4291..4431
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4506..4961
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        5243..5347
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5348..6208
                     /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      6379..6967
                     /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 tagtgcagta gacccaggcc tgccttctgt
     3661 gaagcaagag ccacctgacc cagaggagga caaggaggag aacaaggatg attctgcctc
     3721 caaattggcc ccagaggaag aggcaggagg ggctggcaca cccgtgatca cggagatttt
     3781 cagcctgggt ggaacccgct tccgagatac agcagtctgg ttgccagcta gcggaggtgg
     3841 ttcgccaaag aagaaacgga aggtgtgagc atgcaataaa acatctttgt tttcattaga
     3901 tctgtgtgtt ggttttttgt gtgcggccgg taccgagggc ctatttccca tgattccttc
     3961 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     4021 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     4081 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     4141 cgatttcttg gctttatata tcttgtggaa aggacgaaac accggagacc acggcaggtc
     4201 tcagttttag tccctgaaaa gggactaaaa taaagagttt gcgggactct gcggggttac
     4261 aatcccctaa aaccgctttt ttgcggccgc aggaacccct agtgatggag ttggccactc
     4321 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4381 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4441 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4501 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4561 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4621 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4681 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4741 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4801 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct attcttttga
     4861 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4921 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     4981 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5041 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5101 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     5161 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     5221 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5281 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5341 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5401 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5461 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5521 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5581 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5641 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5701 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5761 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5821 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5881 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5941 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6001 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6061 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6121 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6181 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6241 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6301 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6361 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6421 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6481 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     6541 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6601 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6661 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6721 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6781 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6841 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6901 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6961 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7021 tcacatgt
//
