LOCUS       Exported                3312 bp DNA     circular SYN 05-MAR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3312)
  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..3312
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        39..279
                     /label=U6 promoter
     primer_bind     39..59
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     84..108
                     /label=Anna 52 U6 FW primer
     primer_bind     210..229
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(220..239)
                     /label=oJWseq4-U6 RV
     misc_feature    259..264
                     /label=TATA box
                     /note="RNA transcription starts 23(+1) bases from the end 
                     of this recognition sequence"
     primer_bind     270..323
                     /label=AJ21030
     primer_bind     complement(276..296)
                     /label=AJ21032
     misc_RNA        306..317
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    319..322
                     /label=extension
     protein_bind    complement(323..356)
                     /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    357..360
                     /label=extension
     misc_RNA        361..421
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    422..432
                     /label=Termination sequence
                     /note="RNA polymerase will stop transcription at this 
                     position and fall off"
     primer_bind     complement(480..555)
                     /label=AJ21031
     protein_bind    complement(492..525)
                     /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    526..536
                     /label=termination sequence
     misc_feature    537..583
                     /label=Multiple cloning site
     primer_bind     complement(544..569)
                     /label=AJ20121
     primer_bind     complement(585..604)
                     /label=3REV
     repeat_region   613..753
                     /label=AAV2 ITR
     primer_bind     complement(659..670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     714..731
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      828..1283
                     /direction=RIGHT
                     /label=f1 ori
     primer_bind     complement(915..934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1125..1146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1300..1319)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1419..1441
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1479..1497)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1565..1669
                     /label=AmpR promoter
     CDS             1670..2530
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1888..1907)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2701..3289
                     /direction=RIGHT
                     /label=ori
     primer_bind     3190..3209
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(3303..3312,1..11)
                     /label=20122
ORIGIN
        1 acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc
       61 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      121 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      181 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      241 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa
      301 gacctgtttt agagctatgc tggaagttcc tattctctag aaagtatagg aacttccagc
      361 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      421 cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg
      481 tgcgccaatt cgaagttcct attctctaga aagtatagga acttcttttt tttttttcta
      541 gaagcttgct agcaattgtc gactcgagga tcctaggacg cgttaactta cggtaaatgg
      601 cccggcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
      661 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
      721 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
      781 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
      841 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
      901 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
      961 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1021 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     1081 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1141 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     1201 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1261 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     1321 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     1381 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     1441 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     1501 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     1561 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1621 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1681 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1741 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1801 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1861 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     1921 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1981 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2041 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2101 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2161 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     2221 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2281 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2341 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     2401 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2461 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2521 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2581 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2641 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2701 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2761 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2821 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2881 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2941 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3001 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3061 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3121 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3181 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3241 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3301 cgcggccttt tt
//
