LOCUS       Exported                2872 bp ds-DNA     circular SYN 14-MAR-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    P166_pTGC-U6.2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2872)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Mar 14, 2019 from SnapGene 3.3.2
            http://www.snapgene.com
COMMENT     pDONR221
FEATURES             Location/Qualifiers
     source          1..2872
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      complement(268..295)
                     /note="rrnB T2 transcription termination"
                     /note="rrnB T2 transcription termination sequence"
     terminator      387..473
                     /gene="Escherichia coli rrnB"
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     537..553
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        576..651
                     /note="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      652..695
                     /note="U6:1 terminator"
                     /note="U6:96Aa 3' flanking sequence"
     promoter        696..1095
                     /note="U6:2 promoter"
                     /note="Used as U6:2 promoter in CFD2 gRNA vector and U6b 
                     promotor of Jianquan Ni's and O'Connor-Giles' gRNA vector"
     exon            696..953
                     /note="Esty2 exon"
     promoter        complement(1114..1132)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1137..1153)
                     /note="M13 reverse primer"
     CDS             1266..2075
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      2222..2810
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 tcgggccgct cttctgtttt agagctagaa atagcaagtt
      601 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttgt
      661 cacatctaca atcgttttat atgttctctt ctttcgttcg acttgcagcc tgaaatacgg
      721 cacgagtagg aaaagccgag tcaaatgccg aatgcagagt ctcattacag cacaatcaac
      781 tcaagaaaaa ctcgacactt ttttaccatt tgcacttaaa tcctttttta ttcgttatgt
      841 atactttttt tggtccctaa ccaaaacaaa accaaactct cttagtcgtg cctctatatt
      901 taaaactatc aatttattat agtcaataaa tcgaactgtg ttttcaacaa acgaacaata
      961 ggacactttg attctaaagg aaattttgaa aatcttaagc agagggttct taagaccatt
     1021 tgccaattct tataattctc aactgctctt tcctgatgtt gatcatttat ataggtatgt
     1081 tttcctcaat acttcgggaa gagcctgata tcccctatag tgagtcgtat tacatggtca
     1141 tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag
     1201 ataaaataat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     1261 gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg
     1321 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     1381 atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     1441 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     1501 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     1561 tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     1621 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     1681 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     1741 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     1801 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     1861 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     1921 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     1981 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     2041 tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac
     2101 actggcagag cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta
     2161 acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2221 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2281 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2341 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2401 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2461 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2521 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2581 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2641 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2701 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2761 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2821 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tt
//
