LOCUS       Exported                4311 bp ds-DNA     circular SYN 17-JAN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4311)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Jan 21, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4311
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    10..109
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    152..169
                     /label=MCS
     CDS             182..727
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             743..1051
                     /codon_start=1
                     /label=p53DD
                     /translation="MTAMEESQSDISLKRALPTCTSASPPQKKKPLDGEYFTLKIRGRK
                     RFEMFRELNEALELKDAHATEESGDSRAHSSYLKTKKGQSTSRHKKTMVKKVGPDSD"
     misc_feature    1065..1603
                     /label=IRES
     primer_bind     complement(1208..1225)
                     /label=oSEQ040 IRES_rev
     primer_bind     1435..1454
                     /label=oSEQ039 IRES_fwd
     CDS             1609..2178
                     /codon_start=1
                     /product="human oncoprotein generated by the G12V mutation 
                     in the small GTPase H-Ras"
                     /label=H-Ras (G12V)
                     /translation="MTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVV
                     IDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVK
                     DSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREI
                     RQHKLRKLNPPDESGPGCMSCKCVLS"
     primer_bind     2064..2082
                     /label=oSEQ092 HRas-mid_fwd
     primer_bind     complement(2157..2202)
                     /label=P2A-hRAS-Rev
                     /note="Melting Temp - 66 C"
     primer_bind     complement(2160..2202)
                     /label=P2A-hRAS-Rev-v02
     protein_bind    complement(2192..2291)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2313..2338)
                     /label=oSEQ033 Pentr_rev
     primer_bind     complement(2315..2334)
                     /label=GW_ATTL2
     primer_bind     complement(2327..2348)
                     /label=oSEQ044 pENTR_rev_seq
     primer_bind     complement(2400..2420)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
     CDS             2414..3223
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2439..2460)
                     /label=oSEQ023 attl2_rev
     primer_bind     complement(2447..2470)
                     /label=OSEQ087 Kan_rev_SEQ
     rep_origin      3313..3901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3802..3821
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     4053..4066
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      4066..4152
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4215..4234
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      4244..4271
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     4273..4292
                     /label=oSEQ043 pENTR_fwd_seq
ORIGIN
        1 ctcgggcccc aaataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt
       61 gataagcaat gcttttttat aatgccaact ttgtacaaaa aagcaggctc caccatggga
      121 accaattcag tcgactggat ccggtactac tgaattcaag cttggatccc aatgtgccac
      181 catggacaaa gactgcgaaa tgaagcgcac caccctggat agccctctgg gcaagctgga
      241 actgtctggg tgcgaacagg gcctgcaccg tatcatcttc ctgggcaaag gaacatctgc
      301 cgccgacgcc gtggaagtgc ctgccccagc cgccgtgctg ggcggaccag agccactgat
      361 gcaggccacc gcctggctca acgcctactt tcaccagcct gaggccatcg aggagttccc
      421 tgtgccagcc ctgcaccacc cagtgttcca gcaggagagc tttacccgcc aggtgctgtg
      481 gaaactgctg aaagtggtga agttcggaga ggtcatcagc tacagccacc tggccgccct
      541 ggccggcaat cccgccgcca ccgccgccgt gaaaaccgcc ctgagcggaa atcccgtgcc
      601 cattctgatc ccctgccacc gggtggtgca gggcgacctg gacgtggggg gctacgaggg
      661 cgggctcgcc gtgaaagagt ggctgctggc ccacgagggc cacagactgg gcaagcctgg
      721 gctgggtcct gcaggcggat ccatgactgc catggaggag tcacagtcgg atatcagcct
      781 caagagagcg ctgcccacct gcacaagcgc ctctcccccg caaaagaaaa aaccacttga
      841 tggagagtat ttcaccctca agatccgcgg gcgtaaacgc ttcgagatgt tccgggagct
      901 gaatgaggcc ttagagttaa aggatgccca tgctacagag gagtctggag acagcagggc
      961 tcactccagc tacctgaaga ccaagaaggg ccagtctact tcccgccata aaaaaacaat
     1021 ggtcaagaaa gtggggcctg actcagactg acaacaccat ggagcgttac tggccgaagc
     1081 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     1141 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     1201 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     1261 ctggaagctt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
     1321 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
     1381 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
     1441 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
     1501 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
     1561 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacaggaat gaccgaatac
     1621 aagctggtgg tggtgggcgc cgtcggtgtg ggcaagagtg cgctgaccat ccagctgatc
     1681 cagaaccatt ttgtggacga atacgacccc actatagagg attcctaccg gaagcaggtg
     1741 gtcattgatg gggagacgtg cctgttggac atcctggata ccgccggcca ggaggagtac
     1801 agcgccatgc gggaccagta catgcgcacc ggggagggct tcctgtgtgt gtttgccatc
     1861 aacaacacca agtcttttga ggacatccac cagtacaggg agcagatcaa acgggtgaag
     1921 gactcggatg acgtgcccat ggtgctggtg gggaacaagt gtgacctggc tgcacgcact
     1981 gtggaatctc ggcaggctca ggacctcgcc cgaagctacg gcatccccta catcgaaacc
     2041 tcggccaaga cccggcaggg agtggaggat gccttctaca cgttggtgcg tgagatccgg
     2101 cagcacaagc tgcggaagct gaaccctcct gatgagagtg gccccggctg catgagctgc
     2161 aagtgtgtgc tctcctaacg ctgatatcta gacccagctt tcttgtacaa agttggcatt
     2221 ataagaaagc attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
     2281 atcattattt gccatccagc tgcagctctg gcccgtgtct caaaatctct gatgttacat
     2341 tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa
     2401 tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa
     2461 catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc
     2521 gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa
     2581 aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
     2641 tatgcctctt ccgaccatca agcattttat ccgtactcct ggtgatgcat ggttactcac
     2701 cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga
     2761 aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa
     2821 ttgtcctttt aacagcgatc gcgtatttcg tctagctcag gcgcaatcac gaatgaataa
     2881 cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
     2941 ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
     3001 tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
     3061 acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
     3121 gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
     3181 gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
     3241 gttgtaacat tattcagatt gggccccgtt ccactgagcg tcagaccccg tagaaaagat
     3301 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3361 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3421 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     3481 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3541 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3601 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3661 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3721 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3781 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3841 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3901 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3961 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcta gcatggatct
     4021 cggggacgtc taactactaa gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag
     4081 gctcagtcgg aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     4141 agtaggacaa atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg
     4201 cgggcaggac gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg
     4261 gatggccttt ttgcgtttct acaaactctt cctgttagtt agttacttaa g
//
