LOCUS       sequence-367451-        6450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NMA111 with both nuclear localization signals mutated to alanines 
            fused to GFP (mutagenesis of KBB280).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6450)
  AUTHORS   Belanger KD, Walter D, Henderson TA, Yelton AL, O'Brien TG, Belanger
            KG, Geier SJ, Fahrenkrog B
  TITLE     Nuclear localisation is crucial for the proapoptotic activity of the
            HtrA-like serine protease Nma111p.
  JOURNAL   J Cell Sci. 2009 Nov 1;122(Pt 21):3931-41. doi: 10.1242/jcs.056887. 
            Epub 2009 Oct 6.
  PUBMED    19808885
REFERENCE   2  (bases 1 to 6450)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Sci."; date: "2009-11-1"; volume: "122(Pt 21):3931-41. doi"; pages: 
            " 10.1242/jcs.056887. Epub 2009 Oct 6"
COMMENT     Sequence Label: KBB394
FEATURES             Location/Qualifiers
     source          1..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(400..468,469..1260)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(618..637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1431..2019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1920..1939
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2173..2190
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2307..2328
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2343..2348,2349..2366,2367..2373)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2381..2397
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     2386..2408
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2405..2421
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2405..2421
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2442..2460
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2488..2504
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2489..2505
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3126..3836
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(3153..3181)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3776..3796
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3887..3905)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3901..3948
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(4155..4174)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4156..4174)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4181..4198)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4181..4197)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4190..4212)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4338..4793
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4425..4444)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4635..4656
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4924..5727)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5728..5943)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5967..5986)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6086..6108
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6146..6164)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6205..6450,1..258)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 aacgataagt aaaatgtaaa atcacaggat tttcgtgtgt ggtcttctac acagacaaga
       61 tgaaacaatt cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt
      121 tggcgatccc cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt
      181 tcttttttta ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt
      241 atttttatag cacgtgatga aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga
      301 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
      361 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
      421 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
      481 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
      541 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
      601 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
      661 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
      721 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
      781 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
      841 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
      901 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
      961 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     1021 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     1081 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     1141 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     1201 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     1261 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     1321 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     1381 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1441 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1501 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1561 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1621 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1681 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1741 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1801 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1861 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1921 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1981 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2041 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2101 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2161 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     2221 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     2281 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     2341 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     2401 cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc tcactaaagg
     2461 gaacaaaagc tggtaccggg ccccccctcg aggtcgacgg tatcgataag cttgatatcg
     2521 aattcctgca ggcatcagca tcagcaagat ccaagaacct agagtcacga ttgaaatatt
     2581 taataataaa tgctatgtat catagataaa ctagaaagag gcaagaaaaa tttaacaagt
     2641 ttcttaattg aatattattt tactgtcgct accattatta gacgccatct cagcgtgtga
     2701 catgataatt tttctttttt tctttttttt tcctcaaggc gatgatatga aactttcaaa
     2761 aaatttatat gtaaataaaa aagttctacg agggtttaaa tacggaagtt tatttagaag
     2821 atatttatac tctgaataca cacgtagagt acagtaaagg ttttttagat ctactaatga
     2881 ccatatcgtt gagcaatata gctgctgctg accattctaa aatttccgat ggaacttctg
     2941 gtgaatcatc gcttgtagct gctgctcagt tggaaagcgc caccggagat caagaggaag
     3001 aatatacaga ccatgaaatc attatagagc cgttgcactt tgctaataat aataacactg
     3061 tactcacaga ttcagaaaat tatctaagat ggcaaaatac catctccaac gttgttaaat
     3121 cagtgagcaa aggagaagaa ctcttcactg gagttgtccc aattcttgtt gaattagatg
     3181 gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg
     3241 gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca tggccaacac
     3301 ttgtcactac tttcacttat ggtgttcagt gcttttcaag atacccggat catatgaaac
     3361 ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga actatatttt
     3421 tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt gatacccttg
     3481 ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt cttgggcaca
     3541 aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa caaaagaatg
     3601 gaatcaaagc taacttcaaa attagacaca acattgaaga tggaagcgtt caactagcag
     3661 accattatca acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt
     3721 acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc
     3781 ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaaggat
     3841 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     3901 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     3961 actatatccg gatatccaca ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc
     4021 tccaagtagc gaagcgagca ggactgggcg gcggccaaag cggtcggaca gtgctccgag
     4081 aacgggtgcg catagaaatt gcatcaacgc atatagcgct agagcggccg ccaccgcggt
     4141 ggagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
     4201 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4261 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4321 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     4381 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     4441 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     4501 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4561 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4621 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4681 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4741 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     4801 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagggta ataactgata
     4861 taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt ataatacagt
     4921 tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg
     4981 ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat
     5041 gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc
     5101 cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc
     5161 acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc
     5221 aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca attctgctaa
     5281 catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca aaccgctaac
     5341 aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta
     5401 tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc
     5461 gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat
     5521 ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg gacctaatgc
     5581 ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt
     5641 ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc
     5701 acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct
     5761 gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac atatgcgtat
     5821 atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg agattaccga
     5881 atcaaaaaaa tttcaaggaa accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt
     5941 gaaaaggtgg tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6001 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     6061 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6121 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     6181 atgataataa tggtttctta gacggatcgc ttgcctgtaa cttacacgcg cctcgtatct
     6241 tttaatgatg gaataatttg ggaatttact ctgtgtttat ttatttttat gttttgtatt
     6301 tggattttag aaagtaaata aagaaggtag aagagttacg gaatgaagaa aaaaaaataa
     6361 acaaaggttt aaaaaatttc aacaaaaagc gtactttaca tatatattta ttagacaaga
     6421 aaagcagatt aaatagatat acattcgatt
//
