LOCUS       Exported                5797 bp ds-DNA     circular SYN 04-ABR-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pD092-pUC19 KGA HA5'-mKO2-P2A-Zeo-3'
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 5797)
  AUTHORS   Douglas Adamoski
  TITLE     Direct Submission
  JOURNAL   Exported quarta-feira, 4 de abr de 2018 from SnapGene 2.8.3
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5797
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     CDS             complement(146..406)
                     /codon_start=1
                     /gene="lacZ"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /note="lacZ-alpha"
                     /translation="SSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRP
                     SQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     primer_bind     379..395
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    396..406
                     /note="MCS"
                     /note="pUC19 multiple cloning site"
     misc_feature    408..1397
                     /note="5' Homology Arm"
     intron          408..1243
     CDS             1244..1397
                     /codon_start=1
                     /gene="GLS"
                     /gene_synonym="AAD20; GAC; GAM; GLS1; KGA"
                     /product="glutaminase kidney isoform, mitochondrial isoform
                     1 precursor"
                     /EC_number="3.5.1.2"
                     /note="GLS"
                     /note="isoform 1 precursor is encoded by transcript variant
                     1; glutaminase C; L-glutamine amidohydrolase; 
                     K-glutaminase; glutaminase, phosphate-activated; 
                     glutaminase kidney isoform, mitochondrial"
                     /db_xref="GI:156104878"
                     /db_xref="CCDS:CCDS2308.1"
                     /db_xref="GeneID:2744"
                     /db_xref="HGNC:HGNC:4331"
                     /db_xref="MIM:138280"
                     /protein_id="NP_055720.3"
                     /translation="WNNTPMDEALHFGHHDVFKILQEYQVQYTPQGDSDNGKENQTVHK
                     NLDGLL"
     misc_signal     1381..1397
                     /note="sgRNA"
     CDS             1407..2060
                     /codon_start=1
                     /note="mKO2"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2097..2162
                     /codon_start=1
                     /note="P2A Porcine teschovirus-1"
                     /translation="GSGATNFSLLKQAGDVEENPGP"
     CDS             2169..2543
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MDAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD
                     DVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWG
                     REFALRDPAGNCVHFVAEEQD"
     misc_feature    2550..3539
                     /note="3' Homology Arm"
     misc_signal     2550..2552
                     /note="sgRNA"
     CDS             2550..2552
                     /codon_start=1
                     /gene="GLS"
                     /gene_synonym="AAD20; GAC; GAM; GLS1; KGA"
                     /product="glutaminase kidney isoform, mitochondrial isoform
                     1 precursor"
                     /EC_number="3.5.1.2"
                     /note="GLS"
                     /note="isoform 1 precursor is encoded by transcript variant
                     1; glutaminase C; L-glutamine amidohydrolase; 
                     K-glutaminase; glutaminase, phosphate-activated; 
                     glutaminase kidney isoform, mitochondrial"
                     /db_xref="GI:156104878"
                     /db_xref="CCDS:CCDS2308.1"
                     /db_xref="GeneID:2744"
                     /db_xref="HGNC:HGNC:4331"
                     /db_xref="MIM:138280"
                     /protein_id="NP_055720.3"
                     /translation=""
     misc_feature    2553..2555
                     /note="PAM NGG"
     polyA_signal    2730..2735
                     /gene="GLS"
                     /gene_synonym="AAD20; GAC; GAM; GLS1; KGA"
     polyA_site      2763
                     /gene="GLS"
                     /gene_synonym="AAD20; GAC; GAM; GLS1; KGA"
     variation       3043
                     /note="Mutation (A->T)"
     variation       3045
                     /note="Mutation (C->A)"
     variation       3436
                     /note="Mutation (A->G)"
     CDS             complement(3541..3580)
                     /codon_start=1
                     /gene="lacZ"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /note="lacZ-alpha"
                     /translation="MTMITPSLHACRS"
     misc_feature    3541..3563
                     /note="MCS"
                     /note="pUC19 multiple cloning site"
     primer_bind     complement(3576..3592)
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3600..3616
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(3624..3654)
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     rep_origin      complement(3978..4566)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4737..5597)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5598..5702)
                     /gene="bla"
                     /note="AmpR promoter"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctccag gcttcacagt
      421 ttgtaaagtg taagggaact acccatcgta ccctgtcatt gactagggct gtgagttatg
      481 tagttctgtc tcctcttgca aaagacttac cacttctggc aagtgattaa ccacttctgg
      541 caactcttca tttcttctta tccttgaata ttcatctaca tcactctaaa cagcacagcc
      601 ccagaagcat ggaaagggga gttattagta tggaaagggg agttactctt ctggtgtagt
      661 ggtccgattg agtccatggc ttcccagcct taccagagct gataaaaatg tcaattcctt
      721 tggggccaat cttgctcctc cagtgtgttt tagccctaat gaggtcatgg ttatttctag
      781 acttctgaga cttactgtgg ctttgaattg acacaaacac taattttctg tcaaaggcta
      841 gagtgatgga tgttatatgc ctgaattttc tttgaacatt ttattttaaa agagatactt
      901 tgggcttttc atgaaactaa aattctggaa ccctagagca gtctattata tctgtcacca
      961 caaggacttt ccttaaaaag ctccctccat aagcacttgg atgtgtaaac aagatccttt
     1021 cacagatctt tttctcttcc tctccatatc ccaacttgtc ttggagacta ctttttgaat
     1081 tataaatccc tttctgcatt tctattatta atttttattt gtaaaattgc taaaatagct
     1141 aaatttggcc atttaacctg catttaaaat ctaggaatgt ggggtgatca tctttgtaca
     1201 taaattacct aatgaccctg tccatgctgt gctacgtgtt taggtggaat aacactccca
     1261 tggatgaagc actgcacttt ggacaccatg atgtatttaa aattctccaa gaataccaag
     1321 tccagtacac acctcaagga gattctgaca acgggaagga aaatcaaacc gtccataaga
     1381 atcttgatgg attgttggga tctaccatgg tgagcgtgat caagcccgag atgaagatga
     1441 ggtactacat ggacggctcc gtcaatgggc atgagttcac aatcgagggt gagggcacag
     1501 gcagacctta cgagggacat caggagatga cactgcgcgt cacaatggcc gagggcgggc
     1561 caatgccttt cgccttcgac ctggtgtccc acgtgttctg ttacggccac agagttttta
     1621 ccaagtaccc agaagagatc ccagactatt tcaagcaggc ctttcctgag ggcctgtcct
     1681 gggagaggtc cctggagttc gaggacggcg gctccgcctc cgtgagcgcc cacatcagcc
     1741 tgaggggcaa caccttctac cacaagtcca agttcaccgg cgtgaacttc cccgccgacg
     1801 gccccatcat gcagaaccag agcgtggact gggagccctc caccgagaag atcaccgcca
     1861 gcgacggcgt gctgaagggc gacgtgacca tgtacctgaa gctggagggc ggcggcaacc
     1921 acaagtgcca gatgaagacc acctacaagg ccgccaagga gatcctggag atgcccggcg
     1981 accactacat cggccacagg ctggtgagga agaccgaggg caacatcacc gagcaggtgg
     2041 aggacgccgt ggcccactcc ggtggcggag gctcgggcgg aggtgggtcg ggatcaggaa
     2101 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     2161 ctggatctat ggatgccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     2221 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     2281 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     2341 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     2401 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     2461 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     2521 acttcgtggc cgaggagcag gacggatcct aatggtctca aatcccaaga tttaaatcac
     2581 ttacctattt aattgtggaa aatgattatg aagaacatgt gtatttctat ctggtagtga
     2641 tgtatatttt acatttgtca tttcagtgtt actggagttt tcttcattgt gcacacagga
     2701 caaatctgat ctctttggga aaaaatagaa ataaaacaat ctccctccat aatgtgagca
     2761 atattacctc gtgcattgta taatttgatg taaaagaaat agttaccaat gctagcttgt
     2821 gtggtcttcc atgatttatt tgtgttttgt gaattttcaa tttatggtga tgatctgctg
     2881 atatgcattt ataaagtaag ctctgttgta cagtctgtcc aaatgggtca aggttgcctt
     2941 tagaagcaaa tagtgtgatt ttcaagactt caaatacaaa tttagtttaa gtgtttgaac
     3001 aactatatgc acttacggtt gtgtgtttaa aatgtctctc tctcacccta gcttcatgat
     3061 gtgactctta aaaaactata atagttaaca actgttagta agatagacca attctgatta
     3121 gactttatca gggaatctgt ttaagatatg tttggtgacc aaaacgtatg tgtgaatgta
     3181 gttataatgc ttttgaaaaa ttttcctttt tctatatccc cttagtccag cctctcttct
     3241 cagacattta gctatctgcc tctttccttt agctgggaaa gtgagagctg gcatactatg
     3301 cagtttttat gttttccata gtaagtcaga aaatgcctcc tatttctggc atcagaactt
     3361 tgccatttgt ctacagaaga cgaaccagag acaaaattac taagtataaa ttagtcaagt
     3421 ttatcagtct aaaaagcgaa gggatgtgca actgcagctc tttaagaagt tttttttttt
     3481 tagcttctag ggtaaagata aattcagaaa tgctctaagc taccaaagtt attctgaaag
     3541 tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3601 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3661 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3721 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3781 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3841 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3901 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3961 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4021 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4081 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4141 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4201 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4261 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4321 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4381 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4441 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4501 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4561 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4621 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4681 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4741 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4801 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4861 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4921 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4981 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5041 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5101 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5161 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5221 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5281 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5341 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5401 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5461 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5521 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5581 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5641 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5701 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     5761 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
