LOCUS       Exported                3266 bp ds-DNA     circular SYN 08-AUG-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    LB101_3xHA_bGHpolyA+TRE #2_SaCas9_gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3266)
  AUTHORS   Invitrogen
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Aug 8, 2019 from SnapGene Viewer 3.3.3
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3266
                     /organism="synthetic DNA construct"
                     /note="dnas_title=LB101_3xHA_bGHpolyA+TRE #2_SaCas9_gRNA"
     misc_feature    complement(268..295)
                     /note="rrnB T2 transcription termination sequence"
     misc_feature    complement(427..470)
                     /note="rrnB T1 transcription termination sequence"
     primer_bind     517..533
                     /note="M13 Forward (-40) primer binding site"
     primer_bind     537..552
                     /note="M13 Forward (-20) primer binding site"
     misc_recomb     570..667
                     /note="attL3"
                     /note="attL3 recombination site
                     recognized as attL3"
     misc_feature    665..676
                     /note="flexible linker (GGGS)"
     misc_feature    677..760
                     /note="3x HA epitope tag"
     polyA_signal    788..998
                     /note="bGH Poly A"
                     /note="bGH Poly A
                     
                     "
     promoter        1005..1253
                     /note="human U6 promoter"
     misc_feature    1250..1279
                     /note="gRNA ligation into BsaI"
     misc_feature    1254
                     /note="G required for PolIII TSS"
     misc_feature    1255..1275
                     /note="SaCas9 TRE gRNA #2"
                     /note="SaCas9 TRE gRNA #2
                     gRNA seq: CATCGTTCTCTATCACTGATA "
     misc_RNA        1276..1365
                     /note="SaCas9 sgRNA scaffold"
     misc_feature    1280
                     /note="t=>A eliminates potential terminator sequence"
     misc_feature    1290..1303
                     /note="GAAA=> TGCTGGAAACAGCA enhances interaction with 
                     SaCas9"
     misc_feature    1314..1317
                     /note="taaaa=>taaa to maintain hairpin with other mutation"
     misc_recomb     complement(1379..1476)
                     /note="attL2"
                     /note="attL2 recombination site
                     recognized as attL2"
     misc_feature    1388..658
                     /label=vector
                     /note="pDONR221-P3P2"
                     /note="source=pDONR221-P3P2
                     type=Donor Vector"
     primer_bind     complement(1531..1547)
                     /note="M13 Reverse primer binding site"
     gene            1660..2469
                     /note="Kanamycin resistance gene"
     rep_origin      2590..3263
                     /note="f1 origin"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtataataa
      661 agttggtggc ggatcctacc catacgatgt tccagattac gcttacccat acgatgttcc
      721 agattacgct tacccatacg atgttccaga ttacgcttaa gaattcctag agctcgctga
      781 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
      841 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
      901 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
      961 ggggaggatt gggaagagaa tagcaggcat gctggggagg taccgagggc ctatttccca
     1021 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     1081 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     1141 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     1201 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgcatcgt
     1261 tctctatcac tgatagttta agtactctgt gctggaaaca gcacagaatc tactaaacaa
     1321 ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga tttttgcggc cgcaggaacc
     1381 cagctttctt gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg
     1441 aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgca gggcggccgc
     1501 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     1561 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     1621 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     1681 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1741 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     1801 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1861 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1921 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     1981 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2041 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2101 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2161 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2221 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2281 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2341 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2401 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2461 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     2521 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     2581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2761 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3241 ttttgctggc cttttgctca catgtt
//
