LOCUS       Exported                5517 bp ds-DNA     circular SYN 08-FEB-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5517)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Saturday, Apr 13, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     Imported using the Genbank importer. File name: 
            addgene-plasmid-100902-sequence-195323.gbk SpCas9n (D10A Nickase 
            mutant) with 2A-EGFP and a cloning backbone for 2 custom gRNAs which
            can be cloned in via a one-step reaction. For generation of DSBs 
            with no off-target cuts..
FEATURES             Location/Qualifiers
     source          1..5517
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      362..950
                     /label=ori
     misc_feature    complement(450..474)
                     /label=ME109
     misc_feature    complement(622..641)
                     /label=ME122
     misc_feature    768..787
                     /label=ME206
     primer_bind     851..870
                     /label=pBR322ori-F
     misc_feature    883..903
                     /label=pDG_seq-1
     misc_feature    931..950
                     /label=ME207
     misc_feature    complement(992..1019)
                     /label=QC239
     misc_feature    complement(992..1011)
                     /label=anneal
     misc_feature    1006..1031
                     /label=QC232
     misc_feature    1006..1019
                     /label=USER
     misc_feature    1010..1031
                     /label=aNNEAL
     misc_feature    1012..1510
                     /label=(1) MS2-3'END_gRNA frag
     misc_feature    1019..1336
                     /label=U6 promoter
     primer_bind     1195..1214
                     /label=ACK365
     misc_feature    1259..1278
                     /label=universal U6
     misc_feature    complement(1315..1336)
                     /label=universal reverse
     misc_feature    1338..1357
                     /label=L202S protospacer
     misc_feature    1343..1344
                     /label=L202S
     misc_feature    1343
                     /label=C6 target
     misc_feature    1358..1433
                     /label=sgRNA
     misc_feature    1370..1373
                     /label=TL
     misc_feature    1410..1413
                     /label=SL2
     misc_feature    1434..1435
                     /label=linker
     misc_feature    1436..1458
                     /label=3' MS2 Zalatan
     misc_feature    1438..1476
                     /label=QC125
     misc_feature    complement(1487..1510)
                     /label=a
     misc_feature    complement(1487..1510)
                     /label=QC233
     misc_feature    1507..1510
                     /label=OH-1
     misc_feature    1511..2313
                     /label=CMV frag
     misc_feature    1511..1528
                     /label=anneal
     enhancer        1657..2036
                     /label=CMV enhancer
     misc_feature    1666..1690
                     /label=ACK14
     promoter        2037..2240
                     /label=CMV promoter
     misc_feature    2166..2190
                     /label=ACK15
     misc_feature    2248..2272
                     /label=QC684_IVT-Fwd
     promoter        2282..2300
                     /label=T7 promoter
     misc_feature    complement(2299..2313)
                     /label=anneal
     misc_feature    2310..2313
                     /label=OH-2
     CDS             2314..3030
                     /label=EGFP
     CDS             2314..3030
                     /codon_start=1
                     /label=Translation 2314-3030
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRVEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYSSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    2314..2328
                     /label=QC247 anneal
     misc_feature    2317..2319
                     /label=Inserted V to optimize translation initiation
     misc_feature    2360..2380
                     /label=ME183
     misc_feature    2509..2517
                     /label=Chromophore
     misc_feature    complement(2627..2644)
                     /label=BLR168
     misc_feature    complement(2628..2629)
                     /label=NG PAM
     misc_feature    complement(2630..2649)
                     /label=Protospacer
     misc_feature    2644..2646
                     /label=A>V
     misc_feature    2645..2667
                     /label=BLR167
     misc_feature    complement(2645)
                     /label=Target A
     misc_feature    2646
                     /label=Silent C>A
     misc_feature    complement(2899..2921)
                     /label=QC301
     misc_feature    complement(2899..2915)
                     /label=anneal
     misc_feature    2913..2932
                     /label=L202S protospacer
     misc_feature    2917..2919
                     /label=L202S
     misc_feature    2918
                     /label=C6 target
     misc_feature    2922..2939
                     /label=F
     misc_feature    2923..2939
                     /label=anneal
     misc_feature    2933..2934
                     /label=NG PAM
     misc_feature    2967..2988
                     /label=EGFP-C-For
     misc_feature    3031..3332
                     /label=(2) pDG_polyA frag
     misc_feature    3031..3051
                     /label=QC234
     misc_feature    3031..3034
                     /label=OH-3
     CDS             3031..3033
                     /codon_start=1
                     /label=Translation 3031-3033
                     /translation=""
     misc_feature    complement(3044..3066)
                     /label=QC685_IVT-Rev_bGH
     primer_bind     complement(3052..3069)
                     /label=BGH-rev
     polyA_signal    3058..3265
                     /label=bGH poly(A) signal
     misc_feature    3164..3185
                     /label=pDG_Seq-2
     misc_feature    complement(3204..3228)
                     /label=ACK64
     primer_bind     3274..3290
                     /label=pBluescriptSK
     primer_bind     3274..3290
                     /label=SK primer
     misc_feature    complement(3310..3348)
                     /label=QC235
     misc_feature    complement(3310..3332)
                     /label=a
     misc_feature    3324..3339
                     /label=user opt#2
     misc_feature    3333..3767
                     /label=(3) Com-gRNA frag (QTC223)
     misc_feature    3333..3350
                     /label=QC236
     misc_feature    3333..3348
                     /label=USER
     misc_feature    complement(3334..3352)
                     /label=3' com Zalatan
     misc_feature    complement(3353..3428)
                     /label=sgRNA
     misc_feature    complement(3373..3376)
                     /label=SL2
     misc_feature    complement(3413..3416)
                     /label=TL
     misc_feature    complement(3429..3448)
                     /label=EGFP(V110A) Protospacer
     misc_feature    3443..3445
                     /label=A>V
     misc_feature    complement(3444)
                     /label=Target A
     misc_feature    3449
                     /label=5'G
     misc_feature    complement(3450..3767)
                     /label=U6 promoter
     misc_feature    3450..3471
                     /label=univ. rev
     misc_feature    3450..3471
                     /label=ACK839
     misc_feature    complement(3508..3527)
                     /label=universal U6
     primer_bind     complement(3572..3591)
                     /label=ACK365
     misc_feature    complement(3743..3783)
                     /label=QC237
     misc_feature    complement(3743..3767)
                     /label=anneal
     misc_feature    3767..3799
                     /label=QC238
     misc_feature    3767..3783
                     /label=user
     misc_feature    complement(3768..3784)
                     /label=M13-R
     misc_feature    join(3785..5517,1..1011)
                     /label=(4) pDG_BB
                     
     misc_feature    3785..3801
                     /label=Unique
     misc_feature    3785..3799
                     /label=anneal
     repeat_region   3791..3931
                     /label=AAV2 ITR
     repeat_region   3791..3920
                     /label=AAV2 ITR
     rep_origin      4006..4461
                     /label=f1 ori
     primer_bind     complement(4093..4112)
                     /label=F1ori-R
     misc_feature    4097..4121
                     /label=ME105
     primer_bind     4303..4324
                     /label=F1ori-F
     primer_bind     complement(4478..4497)
                     /label=pRS-marker
     primer_bind     4597..4619
                     /label=pGEX 3'
     primer_bind     complement(4657..4675)
                     /label=pBRforEco
     misc_feature    complement(4691..4715)
                     /label=ME8
     promoter        4743..4847
                     /label=AmpR promoter
     CDS             join(4848..5517,1..191)
                     /label=AmpR
     misc_feature    complement(4943..4967)
                     /label=ACK13
     primer_bind     complement(5066..5085)
                     /label=Amp-R
     misc_feature    complement(5213..5236)
                     /label=ME7
ORIGIN
        1 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
       61 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
      121 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
      181 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
      241 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
      301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
      361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
      421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
      481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
      541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
      601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
      661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
      721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      901 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      961 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tcgctagctg
     1021 tacaaaaaag caggctttaa aggaaccaat tcagtcgact ggatccggta ccaaggtcgg
     1081 gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     1141 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     1201 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     1261 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     1321 gaaaggacga aacaccgcta ttcaagtacc cagtccggtt ttagagctag aaatagcaag
     1381 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgcgcaca
     1441 tgaggatcac ccatgtgctt tttttaagct tgggccgctc gaggtacctc tctacatatg
     1501 acatgtgagc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     1561 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     1621 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     1681 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1741 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     1801 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     1861 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1921 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     1981 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     2041 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     2101 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     2161 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     2221 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagaga tccgcggccg
     2281 ctaatacgac tcactatagg gagagccgcc accatggtga gcaagggcga ggagctgttc
     2341 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2401 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2461 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctatggagtg
     2521 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2581 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2641 cgcgtagagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2701 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2761 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2821 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2881 ggcgacggcc ccgtgctgct gcccgacaac cactattcaa gtacccagtc cgcgctgagc
     2941 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3001 atcactctcg gcatggacga gctgtacaag taactagagc tcgctgatca gcctcgactg
     3061 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3121 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3181 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3241 aagagaatag caggcatgct ggggagcggc cgctctagaa ctagtggatc ccccgggctg
     3301 caggaattcg atacgaatcc tgcaggaaaa aaagatgctc gcaggcattc aggcaccgac
     3361 tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc
     3421 tctaaaacac tacaagaccc gcgtagagcg gtgtttcgtc ctttccacaa gatatataaa
     3481 gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat
     3541 aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact
     3601 aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt
     3661 atatgcaaat atgaaggaat catgggaaat aggccctctt cctgcccgac cttggtaccg
     3721 gatccagtcg actgaattgg ttcctttaaa gcctgctttt ttgtacagtc atagctgttt
     3781 cctgggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3841 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     3901 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     3961 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4021 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4081 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4141 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4201 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4261 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4321 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     4381 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4441 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4501 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4561 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4621 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4681 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4741 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4801 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4861 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4921 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4981 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5041 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5101 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5161 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5221 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5281 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5341 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5401 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5461 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttc
//
