LOCUS       Exported                8743 bp ds-DNA     circular SYN 08-MAR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8743)
  AUTHORS   Macapp
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Mar 9, 2021 from SnapGene Viewer 5.2.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8743
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..130
                     /label=ITR
     misc_feature    126..130
                     /label=LITR
     promoter        143..383
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    412..492
                     /label=saCas9 scaffold
     misc_RNA        412..487
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    413..416
                     /label=F
     misc_feature    417..425
                     /label=harpin1
     misc_feature    426..429
                     /label=stem
     misc_feature    430..440
                     /label=harpin1
     misc_feature    441..444
                     /label=F
     misc_feature    506..761
                     /label=EFS promoter
     promoter        549..760
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    771..776
                     /label=Kozak
     CDS             777..833
                     /codon_start=1
                     /label=BPNLS
                     /translation="MKRTADGSEFESPKKKRKV"
     CDS             813..833
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      840..849
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     misc_feature    849..4007
                     /label=sakkh cas9
     CDS             876..878
                     /label=D10A
     CDS             2517..2519
                     /label=H557A
     CDS             2586..2588
                     /label=N580A
     CDS             3193..3194
                     /label=E782K-aag
     CDS             3750..3751
                     /label=N968K-aag
     CDS             3891..3893
                     /label=R1015H-CAC
     CDS             4008..4034
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4053..4106
                     /codon_start=1
                     /label=BPNLS
                     /translation="KRTADGSEFESPKKKRKV"
     CDS             4086..4106
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    4110..4244
                     /label=SV40_PA_terminator
                     /note="SV40_PA_terminator "
     polyA_signal    4110..4244
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4245..4250
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    4252..5050
                     /label=CBH promoter
     enhancer        4252..4537
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        4539..4816
                     /label=chicken beta-actin promoter
     intron          4817..5045
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             5064..5783
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    5790..5997
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6000..6003
                     /label=U6 Promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     repeat_region   6006..6146
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      6221..6676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6958..7062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7063..7923
                     /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      8094..8682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gagagggcct atttcccatg attccttcat atttgcatat
      181 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
      241 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      301 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      361 tttatatatc ttgtggaaag gacgaaacac cggagaccac ggcaggtctc agttatagta
      421 ctctggaaac agaatctact ataacaaggc aaaatgccgt gtttatctcg tcaacttgtt
      481 ggcgagattt ttgacgcgtc tagactaggt cttgaaagga gtgggaattg gctccggtgc
      541 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
      601 caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
      661 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
      721 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gaccggtcgc gccaccatga
      781 agcggactgc tgatggcagt gaatttgagt ccccaaagaa gaagagaaag gtggggatcg
      841 ccaccatggc caagcggaac tacatcctgg gcctggacat cggcatcacc agcgtgggct
      901 acggcatcat cgactacgag acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag
      961 aggccaacgt ggaaaacaac gagggcaggc ggagcaagag aggcgccaga aggctgaagc
     1021 ggcggaggcg gcatagaatc cagagagtga agaagctgct gttcgactac aacctgctga
     1081 ccgaccacag cgagctgagc ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc
     1141 agaagctgag cgaggaagag ttctctgccg ccctgctgca cctggccaag agaagaggcg
     1201 tgcacaacgt gaacgaggtg gaagaggaca ccggcaacga gctgtccacc aaagagcaga
     1261 tcagccggaa cagcaaggcc ctggaagaga aatacgtggc cgaactgcag ctggaacggc
     1321 tgaagaaaga cggcgaagtg cggggcagca tcaacagatt caagaccagc gactacgtga
     1381 aagaagccaa acagctgctg aaggtgcaga aggcctacca ccagctggac cagagcttca
     1441 tcgacaccta catcgacctg ctggaaaccc ggcggaccta ctatgaggga cctggcgagg
     1501 gcagcccctt cggctggaag gacatcaaag aatggtacga gatgctgatg ggccactgca
     1561 cctacttccc cgaggaactg cggagcgtga agtacgccta caacgccgac ctgtacaacg
     1621 ccctgaacga cctgaacaat ctcgtgatca ccagggacga gaacgagaag ctggaatatt
     1681 acgagaagtt ccagatcatc gagaacgtgt tcaagcagaa gaagaagccc accctgaagc
     1741 agatcgccaa agaaatcctc gtgaacgaag aggatattaa gggctacaga gtgaccagca
     1801 ccggcaagcc cgagttcacc aacctgaagg tgtaccacga catcaaggac attaccgccc
     1861 ggaaagagat tattgagaac gccgagctgc tggatcagat tgccaagatc ctgaccatct
     1921 accagagcag cgaggacatc caggaagaac tgaccaatct gaactccgag ctgacccagg
     1981 aagagatcga gcagatctct aatctgaagg gctataccgg cacccacaac ctgagcctga
     2041 aggccatcaa cctgatcctg gacgagctgt ggcacaccaa cgacaaccag atcgctatct
     2101 tcaaccggct gaagctggtg cccaagaagg tggacctgtc ccagcagaaa gagatcccca
     2161 ccaccctggt ggacgacttc atcctgagcc ccgtcgtgaa gagaagcttc atccagagca
     2221 tcaaagtgat caacgccatc atcaagaagt acggcctgcc caacgacatc attatcgagc
     2281 tggcccgcga gaagaactcc aaggacgccc agaaaatgat caacgagatg cagaagcgga
     2341 accggcagac caacgagcgg atcgaggaaa tcatccggac caccggcaaa gagaacgcca
     2401 agtacctgat cgagaagatc aagctgcacg acatgcagga aggcaagtgc ctgtacagcc
     2461 tggaagccat ccctctggaa gatctgctga acaacccctt caactatgag gtggaccaca
     2521 tcatccccag aagcgtgtcc ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg
     2581 aagaaaacag caagaagggc aaccggaccc cattccagta cctgagcagc agcgacagca
     2641 agatcagcta cgaaaccttc aagaagcaca tcctgaatct ggccaagggc aagggcagaa
     2701 tcagcaagac caagaaagag tatctgctgg aagaacggga catcaacagg ttctccgtgc
     2761 agaaagactt catcaaccgg aacctggtgg ataccagata cgccaccaga ggcctgatga
     2821 acctgctgcg gagctacttc agagtgaaca acctggacgt gaaagtgaag tccatcaatg
     2881 gcggcttcac cagctttctg cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt
     2941 acaagcacca cgccgaggac gccctgatca ttgccaacgc cgatttcatc ttcaaagagt
     3001 ggaagaaact ggacaaggcc aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg
     3061 ccgagagcat gcccgagatc gaaaccgagc aggagtacaa agagatcttc atcacccccc
     3121 accagatcaa gcacattaag gacttcaagg actacaagta cagccaccgg gtggacaaga
     3181 agcctaatag aaagctgatt aacgacaccc tgtactccac ccggaaggac gacaagggca
     3241 acaccctgat cgtgaacaat ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa
     3301 agctgatcaa caagagcccc gaaaagctgc tgatgtacca ccacgacccc cagacctacc
     3361 agaaactgaa gctgattatg gaacagtacg gcgacgagaa gaatcccctg tacaagtact
     3421 acgaggaaac cgggaactac ctgaccaagt actccaaaaa ggacaacggc cccgtgatca
     3481 agaagattaa gtattacggc aacaaactga acgcccatct ggacatcacc gacgactacc
     3541 ccaacagcag aaacaaggtc gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc
     3601 tggacaatgg cgtgtacaag ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa
     3661 actactacga agtgaatagc aagtgctatg aggaagctaa gaagctgaag aagatcagca
     3721 accaggccga gtttatcgcc tccttctaca agaacgatct gatcaagatc aacggcgagc
     3781 tgtatagagt gatcggcgtg aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg
     3841 acatcaccta ccgcgagtac ctggaaaaca tgaacgacaa gaggcccccc cacatcatta
     3901 agacaatcgc ctccaagacc cagagcatta agaagtacag cacagacatt ctgggcaacc
     3961 tgtatgaagt gaaatctaag aagcaccctc agatcatcaa aaagggctat ccctatgacg
     4021 tgcccgatta tgccagcctg ggcagcggct ccaagcgcac cgccgatggt tccgagttcg
     4081 aaagccccaa aaaaaagcgc aaggtctaaa acttgtttat tgcagcttat aatggttaca
     4141 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4201 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcggcggc ccgttacata
     4261 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4321 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     4381 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     4441 cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact ttcctacttg
     4501 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
     4561 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
     4621 attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg
     4681 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
     4741 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
     4801 aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg
     4861 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     4921 gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat
     4981 ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt
     5041 ttcaggttgg accggccgct agcatggtga gcaagggcga ggagctgttc accggggtgg
     5101 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     5161 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     5221 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     5281 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     5341 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     5401 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     5461 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     5521 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     5581 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     5641 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     5701 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     5761 gcatggacga gctgtacaag taaggtaccc tgtgccttct agttgccagc catctgttgt
     5821 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5881 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5941 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     6001 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     6061 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     6121 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     6181 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     6241 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     6301 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     6361 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6421 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     6481 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     6541 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     6601 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     6661 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6721 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6781 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6841 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     6901 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     6961 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     7021 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     7081 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     7141 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     7201 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     7261 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     7321 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     7381 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     7441 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     7501 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     7561 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     7621 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     7681 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     7741 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     7801 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     7861 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7921 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7981 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     8041 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     8101 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     8161 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     8221 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     8281 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8341 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8401 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     8461 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     8521 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     8581 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8641 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8701 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
