LOCUS       pCR8-CDK12(sgR)        7306 bp DNA     circular SYN 07-SEP-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  AUTHORS   FredHutch
  TITLE     Direct Submission
  JOURNAL   Exported Sep 7, 2023 from SnapGene 7.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      268..295
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
     terminator      387..473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     537..553
                     /label=M13-Fwd
                     /note="color: black; sequence: GTAAAACGACGGCCAGT; added: 
                     2022-05-06"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
                     /note="color: #31849b; direction: RIGHT"
     primer_bind     683..701
                     /label=CDK12.TA.Amp-Fwd
                     /note="color: black; sequence: gcaggctgccaccatgccc; added: 
                     2022-05-05"
     misc_feature    690..695
                     /label=Kozak
                     /label=nonstandard type:  Miscellaneous
                     /note="/application_notes=Facilitates translation 
                     initiation ofATG start codon downstream of the Kozak 
                     sequence."
                     /note="/description=Kozak translation initiation sequence"
                     /note="/full_name=Kozak"
                     /note="/official_designation=Kozak"
                     /note="/uuid=5f18b25a1234443494a618363f09f052"
                     /note="color: #800c0c; direction: RIGHT"
     CDS             696..5168
                     /product="Human CDK12. [NM_016507.3]"
                     /label=CDK12
                     /label=hCDK12[NM_016507.3]
                     /note="/official_designation=NM_016507.3"
                     /note="/uuid=6548e19e1b354e1a84d38c4e32fb7b1c"
                     /note="color: #028a02; direction: RIGHT"
     misc_feature    696..3056
                     /label=CDK12 aa1-787
                     /note="Overexpression of this N-term fragment has been 
                     reported to function as a dominant negative mutant.
                     https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446539/
                     "
                     /note="color: #a6acb3"
     primer_bind     934..991
                     /label=sg2mut-Fwd
                     /note="color: black; sequence: 
                     ccgacaccttctccgatgacatggcAttcaaGctagaTcgaCgTgagaacgacgaacg;
                      added: 2022-06-27"
     primer_bind     complement(934..953)
                     /label=sg2mut-Rev
                     /note="color: black; sequence: gtcatcggagaaggtgtcgg; added:
                     2022-06-27"
     misc_feature    955..974
                     /label=sg2
                     /note="color: #ff0000"
     misc_feature    975..977
                     /label=PAM
                     /note="color: #a6acb3"
     primer_bind     1269..1286
                     /label=CDK12.Seq.Fwd1
                     /note="color: black; sequence: tctgggcacaaagaccgg; added: 
                     2022-05-06"
     primer_bind     1941..1960
                     /label=CDK12.Seq.Fwd2
                     /note="color: black; sequence: gatggaaaggagtccaaggg; added:
                     2022-05-06"
     primer_bind     2722..2741
                     /label=CDK12.Seq.Fwd3
                     /note="color: black; sequence: ctggtggagatctgtctccc; added:
                     2022-05-06"
     primer_bind     complement(2820..2870)
                     /label=sg3mut-Rev
                     /note="color: black; sequence: 
                     gtccacacagcgtttcccccaAtcAGAttcCgtttgtcttctttctccata; added:
                     2022-06-27"
     misc_feature    2833..2852
                     /label=sg3
                     /note="color: #ff0000"
     primer_bind     2851..2870
                     /label=sg3mut-Fwd
                     /note="color: black; sequence: gggggaaacgctgtgtggac; added:
                     2022-06-27"
     primer_bind     complement(3236..3264)
                     /label=Mut-REv
                     /note="color: black; sequence: 
                     gcaggaaattctttttgtgacagtattcc; added: 2021-09-14"
     primer_bind     3265..3294
                     /label=Mut-Fwd
                     /note="color: black; sequence: 
                     atcgggctattaagtgttctaacattttgc; added: 2021-09-14"
     misc_feature    3270..3272
                     /label=D859A kinase dead mut amino acid
                     /note="color: #a6acb3"
     primer_bind     3617..3637
                     /label=CDK12.Seq-Fwd4
                     /note="color: black; sequence: gaagaagcaatatcgaaggcg; 
                     added: 2022-07-05"
     primer_bind     3777..3794
                     /label=CDK12-Fwd(qPCR)
                     /note="color: black; sequence: atggctcctccagacctc; added: 
                     2022-02-16"
     primer_bind     complement(3991..4009)
                     /label=CDK12-Rev(qPCR)
                     /note="color: black; sequence: gcaaggcctattgcatccc; added: 
                     2022-02-16"
     primer_bind     complement(5148..5171)
                     /label=CDK12-TA-Rev
                     /note="color: black; sequence: ggtttagtaaggaactcctctccc; 
                     added: 2022-05-05"
     misc_feature    5169..5171
                     /label=3' homology
                     /note="color: #ff0000"
     protein_bind    complement(5185..5284)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
                     /note="color: #31849b; direction: LEFT"
     promoter        complement(5302..5320)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(5325..5341)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     primer_bind     complement(5325..5341)
                     /label=M13-Rev
                     /note="color: black; sequence: CAGGAAACAGCTATGAC; added: 
                     2022-05-06"
     CDS             5772..6563
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /note="color: #ccffcc"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      6656..7244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
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 tgtacaaaaa
      661 agcaggctcc gaattcgccc ttgcaggctg ccaccatgcc caattcagag agacatgggg
      721 gcaagaagga cgggagtgga ggagcttctg gaactttgca gccgtcatcg ggaggcggca
      781 gctctaacag cagagagcgt caccgcttgg tatcgaagca caagcggcat aagtccaaac
      841 actccaaaga catggggttg gtgacccccg aagcagcatc cctgggcaca gttatcaaac
      901 ctttggtgga gtatgatgat atcagctctg attccgacac cttctccgat gacatggcat
      961 tcaagctaga tcgacgtgag aacgacgaac gtcgtggatc agatcggagc gaccgcctgc
     1021 acaaacatcg tcaccaccag cacaggcgtt cccgggactt actaaaagct aaacagaccg
     1081 aaaaagaaaa aagccaagaa gtctccagca agtcgggatc gatgaaggac cggatatcgg
     1141 gaagttcaaa gcgttcgaat gaggagactg atgactatgg gaaggcgcag gtagccaaaa
     1201 gcagcagcaa ggaatccagg tcatccaagc tccacaagga gaagaccagg aaagaacggg
     1261 agctgaagtc tgggcacaaa gaccggagta aaagtcatcg aaaaagggaa acacccaaaa
     1321 gttacaaaac agtggacagc ccaaaacgga gatccaggag cccccacagg aagtggtctg
     1381 acagctccaa acaagatgat agcccctcgg gagcttctta tggccaagat tatgacctta
     1441 gtccctcacg atctcatacc tcgagcaatt atgactccta caagaaaagt cctggaagta
     1501 cctcgagaag gcagtcggtc agtccccctt acaaggagcc ttcggcctac cagtccagca
     1561 cccggtcacc gagcccctac agtaggcgac agagatctgt cagtccctat agcaggagac
     1621 ggtcgtccag ctacgaaaga agtggctctt acagcgggcg atcgcccagt ccctatggtc
     1681 gaaggcggtc cagcagccct ttcctgagca agcggtctct gagtcggagt ccactcccca
     1741 gtaggaaatc catgaagtcc agaagtagaa gtcctgcata ttcaagacat tcatcttctc
     1801 atagtaaaaa gaagagatcc agttcacgca gtcgtcattc cagtatctca cctgtcaggc
     1861 ttccacttaa ttccagtctg ggagctgaac tcagtaggaa aaagaaggaa agagcagctg
     1921 ctgctgctgc agcaaagatg gatggaaagg agtccaaggg ttcacctgta tttttgccta
     1981 gaaaagagaa cagttcagta gaggctaagg attcaggttt ggagtctaaa aagttaccca
     2041 gaagtgtaaa attggaaaaa tctgccccag atactgaact ggtgaatgta acacatctaa
     2101 acacagaggt aaaaaattct tcagatacag ggaaagtaaa gttggatgag aactccgaga
     2161 agcatcttgt taaagatttg aaagcacagg gaacaagaga ctctaaaccc atagcactga
     2221 aagaggagat tgttactcca aaggagacag aaacatcaga aaaggagacc cctccacctc
     2281 ttcccacaat tgcttctccc ccaccccctc taccaactac tacccctcca cctcagacac
     2341 cccctttgcc acctttgcct ccaataccag ctcttccaca gcaaccacct ctgcctcctt
     2401 ctcagccagc atttagtcag gttcctgctt ccagtacttc aactttgccc ccttctactc
     2461 actcaaagac atctgctgtg tcctctcagg caaattctca gccccctgta caggtttctg
     2521 tgaagactca agtatctgta acagctgcta ttccacacct gaaaacttca acgttgcctc
     2581 ctttgcccct cccaccctta ttacctggag atgatgacat ggatagtcca aaagaaactc
     2641 ttccttcaaa acctgtgaag aaagagaagg aacagaggac acgtcactta ctcacagacc
     2701 ttcctctccc tccagagctc cctggtggag atctgtctcc cccagactct ccagaaccaa
     2761 aggcaatcac accacctcag caaccatata aaaagagacc aaaaatttgt tgtcctcgtt
     2821 atggagaaag aagacaaacg gaatctgatt gggggaaacg ctgtgtggac aagtttgaca
     2881 ttattgggat tattggagaa ggaacctatg gccaagtata taaagccaag gacaaagaca
     2941 caggagaact agtggctctg aagaaggtga gactagacaa tgagaaagag ggcttcccaa
     3001 tcacagccat tcgtgaaatc aaaatccttc gtcagttaat ccaccgaagt gttgttaaca
     3061 tgaaggaaat tgtcacagat aaacaagatg cactggattt caagaaggac aaaggtgcct
     3121 tttaccttgt atttgagtat atggaccatg acttaatggg actgctagaa tctggtttgg
     3181 tgcacttttc tgaggaccat atcaagtcgt tcatgaaaca gctaatggaa ggattggaat
     3241 actgtcacaa aaagaatttc ctgcatcggg atattaagtg ttctaacatt ttgctgaata
     3301 acagtgggca aatcaaacta gcagattttg gacttgctcg gctctataac tctgaagaga
     3361 gtcgccctta cacaaacaaa gtcattactt tgtggtaccg acctccagaa ctactgctag
     3421 gagaggaacg ttacacacca gccatagatg tttggagctg tggatgtatt cttggggaac
     3481 tattcacaaa gaagcctatt tttcaagcca atctggaact ggctcagcta gaactgatca
     3541 gccgactttg tggtagccct tgtccagctg tgtggcctga tgttatcaaa ctgccctact
     3601 tcaacaccat gaaaccgaag aagcaatatc gaaggcgtct acgagaagaa ttctctttca
     3661 ttccttctgc agcacttgat ttattggacc acatgctgac actagatcct agtaagcggt
     3721 gcacagctga acagacccta cagagcgact tccttaaaga tgtcgaactc agcaaaatgg
     3781 ctcctccaga cctcccccac tggcaggatt gccatgagtt gtggagtaag aaacggcgac
     3841 gtcagcgaca aagtggtgtt gtagtcgaag agccacctcc atccaaaact tctcgaaaag
     3901 aaactacctc agggacaagt actgagcctg tgaagaacag cagcccagca ccacctcagc
     3961 ctgctcctgg caaggtggag tctggggctg gggatgcaat aggccttgct gacatcacac
     4021 aacagctgaa tcaaagtgaa ttggcagtgt tattaaacct gctgcagagc caaaccgacc
     4081 tgagcatccc tcaaatggca cagctgctta acatccactc caacccagag atgcagcagc
     4141 agctggaagc cctgaaccaa tccatcagtg ccctgacgga agctacttcc cagcagcagg
     4201 actcagagac catggcccca gaggagtctt tgaaggaagc accctctgcc ccagtgatcc
     4261 tgccttcagc agaacagacg acccttgaag cttcaagcac accagctgac atgcagaata
     4321 tattggcagt tctcttgagt cagctgatga aaacccaaga gccagcaggc agtctggagg
     4381 aaaacaacag tgacaagaac agtgggccac aggggccccg aagaactccc acaatgccac
     4441 aggaggaggc agcagcatgt cctcctcaca ttcttccacc agagaagagg ccccctgagc
     4501 cccccggacc tccaccgccg ccacctccac cccctctggt tgaaggcgat ctttccagcg
     4561 ccccccagga gttgaaccca gccgtgacag ccgccttgct gcaactttta tcccagcctg
     4621 aagcagagcc tcctggccac ctgccacatg agcaccaggc cttgagacca atggagtact
     4681 ccacccgacc ccgtccaaac aggacttatg gaaacactga tgggcctgaa acagggttca
     4741 gtgccattga cactgatgaa cgaaactctg gtccagcctt gacagaatcc ttggtccaga
     4801 ccctggtgaa gaacaggacc ttctcaggct ctctgagcca ccttggggag tccagcagtt
     4861 accagggcac agggtcagtg cagtttccag gggaccagga cctccgtttt gccagggtcc
     4921 ccttagcgtt acacccggtg gtcgggcaac cattcctgaa ggctgaggga agcagcaatt
     4981 ctgtggtaca tgcagagacc aaattgcaaa actatgggga gctggggcca ggaaccactg
     5041 gggccagcag ctcaggagca ggccttcact gggggggccc aactcagtct tctgcttatg
     5101 gaaaactcta tcgggggcct acaagagtcc caccaagagg gggaagaggg agaggagttc
     5161 cttactaaac caagggcgaa ttcgacccag ctttcttgta caaagttggc attataaaaa
     5221 ataattgctc atcaatttgt tgcaacgaac aggtcactat cagtcaaaat aaaatcatta
     5281 tttgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
     5341 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     5401 catcatgcct cctctagacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg
     5461 ttgccgggtg acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct
     5521 gttcggttcg taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct
     5581 tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt
     5641 tttttggggt acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc
     5701 gatgtttgat gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa
     5761 agttaaacat tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag
     5821 ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg
     5881 cagtggatgg cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa
     5941 ggcttgatga aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc
     6001 ctggagagag cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca
     6061 ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca
     6121 ttcttgcagg tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa
     6181 aagcaagaga acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg
     6241 ttcctgaaca ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc
     6301 ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg
     6361 cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc
     6421 cggcccagta tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag
     6481 atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga
     6541 tcaccaaggt agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga
     6601 gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6661 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6721 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6781 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6841 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6901 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6961 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7021 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7081 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7141 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7201 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7261 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
