LOCUS       Exported                4666 bp DNA     circular SYN 20-AUG-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4666)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 1, 2022 from SnapGene 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4666
                     /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    580..2014
                     /label=Sequenced 12-8-2021 and 19-8-2021
     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
     misc_feature    796..801
                     /label=Multiple cloning site
     misc_feature    808..830
                     /label=Target
                     /note="Not so efficient but oke"
     primer_bind     821..860
                     /label=oJW373
     CDS             831..863
                     /codon_start=1
                     /label=Linker
                     /translation="GASGSGSSRSG"
     CDS             864..1574
                     /codon_start=1
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(869..886)
                     /label=pEGFP-RP
                     /note="Macrogen Seq primer"
     primer_bind     complement(906..927)
                     /label=EGFP-N
                     /note="Macrogen Seq primer"
     primer_bind     complement(906..922)
                     /label=pEGFP_N
                     /note="Macrogen Seq primer"
     primer_bind     complement(912..927)
                     /label=EGFP-NR
                     /note="Macrogen Seq primer"
     primer_bind     1467..1487
                     /label=EGFP-CF
                     /note="Macrogen Seq primer"
     primer_bind     1514..1535
                     /label=oJWseq11
     misc_feature    1530..1545
                     /label=JWseq24
     primer_bind     1530..1545
                     /label=oJWseq24 GFP C-FW seq primer
     primer_bind     complement(1550..1566)
                     /label=EGFP-CR
                     /note="Macrogen Seq primer"
     primer_bind     complement(1554..1592)
                     /label=oJW374
     primer_bind     1566..1600
                     /label=oJW375
     CDS             1575..1601
                     /codon_start=1
                     /label=Linker
                     /translation="SASGGGSYV"
     CDS             1602..1883
                     /codon_start=1
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     primer_bind     complement(1858..1908)
                     /label=oJW376
     misc_feature    1894..1916
                     /label=Target
                     /note="Not so efficient but oke"
     primer_bind     1918..1960
                     /label=WD0120 Cas9-free OC1 FW
     primer_bind     complement(1922..1964)
                     /label=WD0121 Cas9-free pOC1 RV
     primer_bind     complement(1939..1958)
                     /label=WDseq02 KI_RV
     repeat_region   1967..2107
                     /label=AAV2 ITR
     repeat_region   1967..2096
                     /label=AAV2 ITR
     rep_origin      2182..2637
                     /direction=RIGHT
                     /label=f1 ori
     primer_bind     complement(2269..2288)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2479..2500
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2654..2673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2773..2795
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2833..2851)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2919..3023
                     /label=AmpR promoter
     CDS             3024..3884
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3242..3261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4055..4643
                     /direction=RIGHT
                     /label=ori
     primer_bind     4544..4563
                     /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 gtagcggctc cagtagatct ggggtgagca agggcgagga gctgttcacc ggggtggtgc
      901 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      961 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     1021 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     1081 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     1141 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     1201 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     1261 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     1321 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     1381 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     1441 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     1501 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     1561 tggacgagct gtacagcgct tctggtggtg gtagctacgt aatcctctgg catgagatgt
     1621 ggcatgaagg cctggaagag gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca
     1681 tgtttgaggt gctggagccc ttgcatgcta tgatggaacg gggcccccag actctgaagg
     1741 aaacatcctt taatcaggcc tatggtcgag atttaatgga ggcccaagag tggtgcagga
     1801 agtacatgaa atcagggaat gtcaaggacc tcctccaagc ctgggacctc tattatcatg
     1861 tgttccgacg aatctcaaag tgagtcgact gctcccgtta caatcctgtg gctcccacgc
     1921 gtggatccta ggaccggtcg ggccatttac cgtaagttgc ggccgcagga acccctagtg
     1981 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     2041 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     2101 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2161 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2221 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     2281 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2341 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     2401 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2461 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     2521 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     2581 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     2641 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     2701 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2761 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2821 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2881 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2941 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3001 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3061 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3121 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3181 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3241 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3301 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3361 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3421 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3481 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3541 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3601 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3661 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3721 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     3781 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3841 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3901 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3961 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4021 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4081 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4141 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4201 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4261 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4321 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4381 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4441 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4501 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4561 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4621 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc cttttt
//
