LOCUS       Exported                4766 bp DNA     circular SYN 07-DEC-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4766)
  AUTHORS   Arthur de Jong
  TITLE     Direct Submission
  JOURNAL   Exported Mar 1, 2022 from SnapGene 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4766
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     primer_bind     17..89
                     /label=20124
     primer_bind     25..84
                     /label=20118
     protein_bind    complement(39..72)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence (TTTCTAGA)."
     misc_feature    73..645
                     /label=mU6*
                     /note="see Tiscornia PNAS 
                     2004https://www.pnas.org/content/101/19/7347
                     "
     protein_bind    343..376
                     /label=loxP511
     misc_feature    385..399
                     /label=termination sequence
     primer_bind     392..420
                     /label=AJ20127
     misc_feature    406..594
                     /label=stuffer sequence
                     /note="See Tiscorina et al, PNAS 2004"
     primer_bind     complement(409..431)
                     /label=AJ20126
     protein_bind    607..640
                     /label=loxP511
     misc_feature    651..670
                     /label=CACNA1E target sequence
     misc_RNA        671..682
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    684..687
                     /label=extension
     protein_bind    complement(688..721)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence (TTTCTAGA)."
     primer_bind     706..757
                     /label=AJ20120
     misc_feature    722..725
                     /label=extension
     misc_RNA        726..786
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    787..797
                     /label=Termination sequence
                     /note="RNA polymerase will stop transcription at this 
                     position and fall off"
     misc_feature    871..876
                     /label=Multiple cloning site
     misc_feature    882..884
                     /label=PAM
     misc_feature    885..904
                     /label=target sequence
     primer_bind     908..930
                     /label=AJ20110
     CDS             908..912
                     /codon_start=1
                     /label=Linker w/ NheI
                     /translation="A"
     CDS             923..1969
                     /codon_start=1
                     /label=smFP HA
                     /translation="MYPYDVPDYAGYPYDVPDYAGYPYDVPDYAGGVSKGEELFTGVVP
                     ILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLGGGVQCFS
                     RYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFK
                     EDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEGGYPYDVPDYAGGYPYDV
                     PDYAGGGGYPYDVPDYAGGYPYDVPDYAGGDGSVQLADHYQQNTPIGDGPVLLPDNHYL
                     STQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGYPYDVPDYAGYPYDVPDYAG
                     YPYDVPDYA"
     CDS             926..952
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             956..982
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             986..1012
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1541..1567
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1574..1600
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1613..1639
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1646..1672
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1883..1909
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1913..1939
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1943..1969
                     /codon_start=1
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1959..1984)
                     /label=AJ20111
     CDS             1982..1984
                     /codon_start=1
                     /label=Linker w/ AfeI
                     /translation="A"
     misc_feature    1989..1991
                     /label=PAM
     misc_feature    1992..2011
                     
     misc_feature    2013..2037
                     /label=Multiple cloning site
     primer_bind     complement(2039..2058)
                     /label=3REV
     repeat_region   2067..2207
                     /label=AAV2 ITR
     primer_bind     complement(2113..2124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2168..2185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      2282..2737
                     /direction=RIGHT
                     /label=f1 ori
     primer_bind     complement(2369..2388)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2579..2600
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2754..2773)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2873..2895
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2933..2951)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3019..3123
                     /label=AmpR promoter
     CDS             3124..3984
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3342..3361)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4155..4743
                     /direction=RIGHT
                     /label=ori
     primer_bind     4644..4663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga agttcctatt ctctagaaag
       61 tataggaact tcgatccgac gccgccatct ctaggcccgc gccggccccc tcgcacagac
      121 ttgtgggaga agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt
      181 aactatagag gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta
      241 cattttacat gataggcttg gatttctata agagatacaa atactaaatt attattttaa
      301 aaaacagcac actcgagaac tcaccctaac tgtaaagtta gtataacttc gtatagtata
      361 aattatacga agttataagc cttgtttttt gcgatttttg aattcgttcc tcagaggaac
      421 tgacaagcac cctaacatcc tattggaggc tcactcacgt tttttctatt ttgtttcttg
      481 acagcagagc tcgttgctca ctgtatagct caggttggcc tgacactgat gaggttctcc
      541 agtgactgcc tctacctacc tactgggatg acagaggtgt accaccaagc cacgcccggg
      601 ggatccataa cttcgtatag tataaattat acgaagttat aagccttgtg caggatggct
      661 cgcttcgggg gttttagagc tatgctggaa gttcctattc tctagaaagt ataggaactt
      721 ccagcatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
      781 cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta
      841 gcgcgtgcgc caattctgca gacaaatggc tctagaagct tcccccccga agcgagccat
      901 cctgatggct agcggagcca ccatgtaccc ttatgatgtg cccgattatg ctggctaccc
      961 ttatgatgtt cctgattacg ccggatatcc gtatgacgtg ccagactacg cgggaggtgt
     1021 gagcaaggga gaggagttgt ttaccggcgt ggtccccata ctggtggagt tggatggcga
     1081 cgttaatgga cataaattct cggtccgcgg cgagggagag ggagacgcca ccaatggcaa
     1141 gctgacgctt aagttcattt gcacgactgg aaaattgcct gtcccctggc cgaccctggt
     1201 caccacactg ggcggaggag tgcagtgctt ctcccgttac ccagaccaca tgaagcagca
     1261 cgatttcttc aagagcgcaa tgccggaggg atacgtgcaa gaacggacta tttccttcaa
     1321 ggatgacgga acctataaga cccgtgcgga ggtcaaattc gagggtgaca ccctggtgaa
     1381 ccgaattgaa ctcaaaggaa tcgatttcaa ggaggatgga aatattctgg gtcacaagct
     1441 ggaatacaac ttcaacagcc ataatgtgta cattacggct gataagcaga agaacggcat
     1501 taaggccaat ttcaagatcc gccacaacgt tgagggtgga tatccctacg acgtgcccga
     1561 ttatgcgggc ggatatccgt atgatgttcc agattatgct ggtggtggag gctatcccta
     1621 tgatgtcccc gactacgccg gaggttaccc atacgacgtg cccgactacg ctggaggcga
     1681 tggcagcgtg cagctggcag atcattatca acagaatacc cccataggtg atggccccgt
     1741 tctgcttcca gataatcact acctttccac ccagagcgtg ctttcgaaag acccgaacga
     1801 aaagcgtgat cacatggtcc tgctggagtt tgtgaccgcg gctggaatca ccctgggtat
     1861 ggacgaactc tacaagggtg gctaccccta cgatgtgccg gattacgctg gatatccgta
     1921 tgacgtaccg gactatgccg gttacccgta cgatgtcccg gactacgctt caaccggtag
     1981 cgctgtttcc cccccgaagc gagccatcct gctcgactcg aggatcctag gacgcgttaa
     2041 cttacggtaa atggcccggc ggccgcagga acccctagtg atggagttgg ccactccctc
     2101 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     2161 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     2221 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     2281 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     2341 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     2401 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     2461 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     2521 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     2581 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     2641 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     2701 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     2761 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     2821 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2881 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     2941 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3001 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3061 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3121 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3181 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3241 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3301 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3361 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3421 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3481 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3541 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3601 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3661 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3721 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3781 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga
     3841 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3901 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3961 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4021 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4081 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4141 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4201 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4261 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4321 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4381 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4441 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4501 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4561 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4621 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4681 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4741 aaaaacgcca gcaacgcggc cttttt
//
