LOCUS       Exported                5513 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 5513)
  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..5513
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    complement(223..247)
                     /label=ACK12
     rep_origin      659..1247
                     /label=ori
     misc_feature    complement(722..746)
                     /label=ACK11
     misc_feature    1065..1084
                     /label=ME206
     primer_bind     1148..1167
                     /label=pBR322ori-F
     misc_feature    1228..1247
                     /label=ME207
     misc_feature    complement(1289..1316)
                     /label=QC239
     misc_feature    complement(1289..1308)
                     /label=anneal
     misc_feature    1303..1328
                     /label=QC232
     misc_feature    1303..1316
                     /label=USER
     misc_feature    1307..1328
                     /label=aNNEAL
     misc_feature    1309..1802
                     /label=(1) MS2-SL3_gRNA frag
     misc_feature    1316..1633
                     /label=U6 promoter
     primer_bind     1492..1511
                     /label=ACK365
     misc_feature    1556..1575
                     /label=universal U6
     misc_feature    complement(1612..1633)
                     /label=universal reverse
     misc_feature    1635..1654
                     /label=L202S protospacer
     misc_feature    1640..1641
                     /label=L202S
     misc_feature    1640
                     /label=C6 target
     misc_feature    1655..1750
                     /label=sgRNA
     misc_feature    1667..1670
                     /label=TL
     misc_feature    1707..1710
                     /label=SL2
     misc_feature    1722..1744
                     /label=MS2-SL insert
     misc_feature    1724..1742
                     /label=MS2 (C-var)
     misc_feature    1736..1765
                     /label=QC177
     misc_feature    complement(1779..1802)
                     /label=a
     misc_feature    complement(1779..1802)
                     /label=QC233
     misc_feature    1799..1802
                     /label=OH-1
     misc_feature    1803..2605
                     /label=CMV frag
     misc_feature    1803..1820
                     /label=anneal
     enhancer        1949..2328
                     /label=CMV enhancer
     misc_feature    1958..1982
                     /label=ACK14
     promoter        2329..2532
                     /label=CMV promoter
     misc_feature    2458..2482
                     /label=ACK15
     promoter        2574..2592
                     /label=T7 promoter
     misc_feature    complement(2591..2605)
                     /label=anneal
     misc_feature    2602..2605
                     /label=OH-2
     CDS             2606..3322
                     /label=EGFP
     CDS             2606..3322
                     /codon_start=1
                     /label=Translation 2606-3322
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRVEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYSSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    2606..2620
                     /label=QC247 anneal
     misc_feature    2609..2611
                     /label=Inserted V to optimize translation initiation
     misc_feature    2652..2672
                     /label=ME183
     misc_feature    2801..2809
                     /label=Chromophore
     misc_feature    complement(2919..2936)
                     /label=BLR168
     misc_feature    complement(2920..2921)
                     /label=NG PAM
     misc_feature    complement(2922..2941)
                     /label=Protospacer
     misc_feature    2936..2938
                     /label=A>V
     misc_feature    2937..2959
                     /label=BLR167
     misc_feature    complement(2937)
                     /label=Target A
     misc_feature    2938
                     /label=Silent C>A
     misc_feature    complement(3191..3213)
                     /label=QC301
     misc_feature    complement(3191..3207)
                     /label=anneal
     misc_feature    3205..3224
                     /label=L202S protospacer
     misc_feature    3209..3211
                     /label=L202S
     misc_feature    3210
                     /label=C6 target
     misc_feature    3214..3231
                     /label=F
     misc_feature    3215..3231
                     /label=anneal
     misc_feature    3225..3226
                     /label=NG PAM
     misc_feature    3259..3280
                     /label=EGFP-C-For
     misc_feature    3323..3624
                     /label=(2) pDG_polyA frag
     misc_feature    3323..3343
                     /label=QC234
     misc_feature    3323..3326
                     /label=OH-3
     CDS             3323..3325
                     /codon_start=1
                     /label=Translation 3323-3325
                     /translation=""
     primer_bind     complement(3344..3361)
                     /label=BGH-rev
     polyA_signal    3350..3557
                     /label=bGH poly(A) signal
     misc_feature    complement(3496..3520)
                     /label=ACK64
     primer_bind     3566..3582
                     /label=SK primer
     primer_bind     3566..3582
                     /label=pBluescriptSK
     misc_feature    complement(3602..3640)
                     /label=QC235
     misc_feature    complement(3602..3624)
                     /label=a
     misc_feature    3616..3631
                     /label=user opt#2
     misc_feature    3625..4059
                     /label=(3) Com-gRNA frag (QTC223)
     misc_feature    3625..3642
                     /label=QC236
     misc_feature    3625..3640
                     /label=USER
     misc_feature    complement(3626..3644)
                     /label=3' com Zalatan
     misc_feature    complement(3645..3720)
                     /label=sgRNA
     misc_feature    complement(3665..3668)
                     /label=SL2
     misc_feature    complement(3705..3708)
                     /label=TL
     misc_feature    complement(3721..3740)
                     /label=EGFP(V110A) Protospacer
     misc_feature    3735..3737
                     /label=A>V
     misc_feature    complement(3736)
                     /label=Target A
     misc_feature    3741
                     /label=5'G
     misc_feature    complement(3742..4059)
                     /label=U6 promoter
     misc_feature    3742..3763
                     /label=ACK839
     misc_feature    3742..3763
                     /label=univ. rev
     misc_feature    complement(3800..3819)
                     /label=universal U6
     primer_bind     complement(3864..3883)
                     /label=ACK365
     misc_feature    complement(4035..4075)
                     /label=QC237
     misc_feature    complement(4035..4059)
                     /label=anneal
     misc_feature    4059..4091
                     /label=QC238
     misc_feature    4059..4075
                     /label=user
     misc_feature    complement(4060..4076)
                     /label=M13-R
     misc_feature    join(4077..5513,1..1308)
                     /label=(4) pDG_BB
                     
     misc_feature    4077..4093
                     /label=Unique
     misc_feature    4077..4091
                     /label=anneal
     repeat_region   4083..4223
                     /label=AAV2 ITR
     repeat_region   4083..4212
                     /label=AAV2 ITR
     rep_origin      4298..4754
                     /label=f1 ori
     primer_bind     complement(4385..4404)
                     /label=F1ori-R
     misc_feature    4389..4413
                     /label=ME106
     misc_feature    4436
                     /label=C insertion
     primer_bind     4596..4617
                     /label=F1ori-F
     primer_bind     complement(4771..4790)
                     /label=pRS-marker
     primer_bind     4890..4912
                     /label=pGEX 3'
     primer_bind     complement(4950..4968)
                     /label=pBRforEco
     promoter        5036..5140
                     /label=AmpR promoter
     CDS             join(5141..5513,1..488)
                     /label=AmpR
     misc_feature    complement(5236..5260)
                     /label=ACK13
     primer_bind     complement(5359..5378)
                     /label=Amp-R
ORIGIN
        1 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
       61 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      121 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
      181 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      241 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      301 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
      361 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
      421 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
      481 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
      541 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
      601 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
      661 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
      721 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
      781 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
      841 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      901 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      961 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1021 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1081 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1141 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1201 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1261 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcg ctagctgtac
     1321 aaaaaagcag gctttaaagg aaccaattca gtcgactgga tccggtacca aggtcgggca
     1381 ggaagagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1441 agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1501 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     1561 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     1621 aggacgaaac accgctattc aagtacccag tccggtttta gagctagaaa tagcaagtta
     1681 aaataaggct agtccgttat caacttgaaa aagtggcacc ggcacatgag gatcacccat
     1741 gtgccggtgc tttttttaag cttgggccgc tcgaggtacc tctctacata tgacatgtga
     1801 gctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     1861 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     1921 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     1981 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     2041 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     2101 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     2161 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     2221 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     2281 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     2341 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     2401 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     2461 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     2521 ataagcagag ctggtttagt gaaccgtcag atccgctaga gatccgcggc cgctaatacg
     2581 actcactata gggagagccg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2641 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2701 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2761 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctatggag tgcagtgctt
     2821 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2881 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgtaga
     2941 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     3001 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     3061 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     3121 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     3181 ccccgtgctg ctgcccgaca accactattc aagtacccag tccgcgctga gcaaagaccc
     3241 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     3301 cggcatggac gagctgtaca agtaactaga gctcgctgat cagcctcgac tgtgccttct
     3361 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3421 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3481 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     3541 agcaggcatg ctggggagcg gccgctctag aactagtgga tcccccgggc tgcaggaatt
     3601 cgatacgaat cctgcaggaa aaaaagatgc tcgcaggcat tcaggcaccg actcggtgcc
     3661 actttttcaa gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac
     3721 actacaagac ccgcgtagag cggtgtttcg tcctttccac aagatatata aagccaagaa
     3781 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     3841 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     3901 tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa
     3961 atatgaagga atcatgggaa ataggccctc ttcctgcccg accttggtac cggatccagt
     4021 cgactgaatt ggttccttta aagcctgctt ttttgtacag tcatagctgt ttcctgggcc
     4081 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4141 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     4201 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     4261 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     4321 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     4381 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tcccccgtca
     4441 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4501 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4561 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4621 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4681 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4741 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4801 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4861 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4921 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4981 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5041 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5101 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5161 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5221 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5281 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5341 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5401 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5461 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg cca
//
