LOCUS       Exported                3649 bp DNA     circular SYN 02-OCT-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3649)
  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..3649
                     /organism="synthetic DNA construct"
                     /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
     primer_bind     complement(663..728)
                     /label=20125
     primer_bind     complement(669..728)
                     /label=AJ20119
     misc_RNA        674..685
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    687..690
                     /label=extension
     protein_bind    complement(691..724)
                     /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     709..760
                     /label=AJ20120
     misc_feature    725..728
                     /label=extension
     misc_RNA        729..789
                     /label=gRNA scaffold
                     /note="in RNA form binds to Cas9"
     misc_feature    790..800
                     /label=Termination sequence
                     /note="RNA polymerase will stop transcription at this 
                     position and fall off"
     misc_feature    874..920
                     /label=Multiple cloning site
     primer_bind     complement(881..906)
                     /label=AJ20121
     primer_bind     complement(922..941)
                     /label=3REV
     repeat_region   950..1090
                     /label=AAV2 ITR
     primer_bind     complement(996..1007)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1051..1068
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      1165..1620
                     /direction=RIGHT
                     /label=f1 ori
     primer_bind     complement(1252..1271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1462..1483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1637..1656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1756..1778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1816..1834)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1902..2006
                     /label=AmpR promoter
     CDS             2007..2867
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2225..2244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3038..3626
                     /direction=RIGHT
                     /label=ori
     primer_bind     3527..3546
                     /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 aagccttgtt tgtcttcgtg
      661 tacaagaaga cctgttttag agctatgctg gaagttccta ttctctagaa agtataggaa
      721 cttccagcat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      781 agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt
      841 ttagcgcgtg cgccaattct gcagacaaat ggctctagaa gcttgctagc aattgtcgac
      901 tcgaggatcc taggacgcgt taacttacgg taaatggccc ggcggccgca ggaaccccta
      961 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     1021 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     1081 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     1141 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1201 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     1261 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1321 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1381 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1441 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     1501 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     1561 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     1621 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     1681 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     1741 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     1801 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     1861 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1921 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1981 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2041 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2101 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2161 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2221 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2281 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2341 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2401 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2461 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2521 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     2581 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2641 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2701 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     2761 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2821 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2881 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2941 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3001 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3061 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3181 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttt
//
