LOCUS       Exported                3693 bp DNA     circular SYN 22-MAR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3693)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 1, 2022 from SnapGene 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3693
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     primer_bind     23..51
                     /label=19096
     misc_feature    39..611
                     /label=mU6*
                     /note="see Tiscornia PNAS 
                     2004https://www.pnas.org/content/101/19/7347
                     "
     protein_bind    309..342
                     /label=loxP511
     misc_feature    351..365
                     /label=termination sequence
     misc_feature    372..560
                     /label=stuffer sequence
                     /note="See Tiscorina et al, PNAS 2004"
     primer_bind     complement(568..615)
                     /label=19098
     protein_bind    573..606
                     /label=loxP511
     misc_feature    587..592
                     /label=TATA
     misc_feature    593..615
                     /label=Tata + 23
     misc_feature    616..635
                     /label=Gria1 target sequence
     misc_RNA        636..711
                     /label=gRNA scaffold
     misc_feature    712..722
                     /label=Termination sequence
     primer_bind     740..764
                     /label=WDseq05 KI_FW
     misc_feature    796..801
                     /label=Multiple cloning site
     misc_feature    808..830
                     /label=Target
                     /note="Not so efficient but oke"
     misc_feature    809..1036
                     /label=sequenced 27-2-2021
     CDS             831..838
                     /codon_start=1
                     /label=Linker
                     /translation="GA"
     primer_bind     complement(839..871)
                     /label=HA repair REV AJ18224
     misc_feature    839..842
                     /label=linker with NheI
     CDS             843..869
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(844..867)
                     /label=oJWseq15 HA RV
     CDS             882..908
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    909..913
                     /label=Linker with HpaI
     CDS             914..917
                     /codon_start=1
                     /label=Linker w/ Afe1
                     /translation="K"
     CDS             918..920
                     /codon_start=1
                     /label=Linker w/ AfeI
                     /translation="A"
     misc_feature    921..943
                     /label=Target
                     /note="Not so efficient but oke"
     primer_bind     945..987
                     /label=WD0120 Cas9-free OC1 FW
     primer_bind     complement(949..991)
                     /label=WD0121 Cas9-free pOC1 RV
     primer_bind     complement(966..985)
                     /label=WDseq02 KI_RV
     repeat_region   994..1134
                     /label=AAV2 ITR
     repeat_region   994..1123
                     /label=AAV2 ITR
     primer_bind     complement(994..1013)
                     /label=WDseq06 KI_RV_#2
     primer_bind     complement(1040..1051)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1095..1112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      1209..1664
                     /direction=RIGHT
                     /label=f1 ori
     primer_bind     complement(1296..1315)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1506..1527
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1681..1700)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1800..1822
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1860..1878)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1946..2050
                     /label=AmpR promoter
     CDS             2051..2911
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2269..2288)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3082..3670
                     /direction=RIGHT
                     /label=ori
     primer_bind     3571..3590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga tccgacgccg ccatctctag
       61 gcccgcgccg gccccctcgc acagacttgt gggagaagct cggctactcc cctgccccgg
      121 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
      181 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataagag
      241 atacaaatac taaattatta ttttaaaaaa cagcacactc gagaactcac cctaactgta
      301 aagttagtat aacttcgtat agtataaatt atacgaagtt ataagccttg ttttttgcga
      361 tttttgaatt cgttcctcag aggaactgac aagcacccta acatcctatt ggaggctcac
      421 tcacgttttt tctattttgt ttcttgacag cagagctcgt tgctcactgt atagctcagg
      481 ttggcctgac actgatgagg ttctccagtg actgcctcta cctacctact gggatgacag
      541 aggtgtacca ccaagccacg cccgggggat ccataacttc gtatagtata aattatacga
      601 agttataagc cttgtgggag ccacaggatt gtaacgtttt agagctagaa atagcaagtt
      661 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt
      721 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
      781 ctgcagacaa atggctctag aagcttaccc gttacaatcc tgtggctccc ggcgctagcg
      841 gttacccata cgatgttcca gattacgctg gatctggttc gtatccttat gacgtgcctg
      901 actacgcagg tggaagcgct cccgttacaa tcctgtggct cccacgcgtg gatcctagga
      961 ccggtcgggc catttaccgt aagttgcggc cgcaggaacc cctagtgatg gagttggcca
     1021 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     1081 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     1141 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     1201 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1261 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     1321 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1381 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     1441 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1501 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     1561 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     1621 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     1681 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     1741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     1801 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     1861 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     1921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     2161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     2221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     2281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     2341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     2401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     2461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     2521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     2581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     2641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     2701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     2761 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     2821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     2881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     2941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     3361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     3421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     3481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     3541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     3601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     3661 cctatggaaa aacgccagca acgcggcctt ttt
//
