LOCUS       sequence-238933-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CEN LEU2 CNB1mutEF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  AUTHORS   Connolly S, Quasi-Woode D, Waldron L, Eberly C, Waters K, Muller EM,
            Kingsbury TJ
  TITLE     Calcineurin Regulatory Subunit Calcium-Binding Domains 
            Differentially Contribute to Calcineurin Signaling in Saccharomyces 
            cerevisiae.
  JOURNAL   Genetics. 2018 Jul;209(3):801-813. doi: 10.1534/genetics.118.300911.
            Epub 2018 May 7.
  PUBMED    29735720
REFERENCE   2  (bases 1 to 7074)
  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"; doi: 
            "10.1534/genetics.118.300911"; journalName: "Genetics"; date: 
            "2018-07"; volume: "209"; issue: "3"; pages: "801-813"
COMMENT     Sequence Label: pTJK513
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     75..91
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     75..91
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1193..1209)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1194..1210)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1238..1256)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1277..1293)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1277..1293)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1290..1312)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1301..1317
                     /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)."
     promoter        complement(join(1325..1331,1332..1349,1350..1355))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1370..1391
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1508..1525)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1679..2267)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1759..1778)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2438..3229,3230..3298))
                     /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     3061..3080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3299..3403)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3440..3943
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3985..4003
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4041..4063)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4163..4182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(4676..5770)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(5771..6178)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(6479..6934)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6616..6637)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6828..6847
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(7060..7074,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(7074,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg gagctccacc
       61 gcggtggcgg ccgctctaga actagtggat ccacgctatg aaaaaggaaa acaaatgact
      121 cttataattt gtttccctcg acttctctat ttaaatccaa gattttaact aataaactat
      181 ttaataataa aacaattaac tctttatatg gaagccttgg aaattagccg ccaaaatggg
      241 atatacattc cgtgcgaagt gaccgcgtgg aaggccgggt atcatcttaa aaatcactag
      301 tttctttttt agcggaatgc aataaaggtg ctttgtgctg gtgtggttta cacggaacat
      361 ctagtagcta aaacttggta actcaatggt gatcagaatc catagaagca tttttatttc
      421 ttaaaatggg tgctgctcct tccaaaattg tggatggtct tttagaagat acaaattgta
      481 tgtacacttc ggagtgagga aaagaaagaa aggggaaatt aaccgatttt actaacactg
      541 acactttgaa cagttgatag agatgaaatt gaaaggttaa ggaagagatt catgaaatta
      601 gctagagata gctcagggtc tattgataaa aatgaattta tgagcattcc tggcgtttcg
      661 tcaaaccctc ttgctggacg tataatggag gttttcgatg ctgataatag tggggacgtg
      721 gattttcaag agttcatcac aggattatcc attttcagtg ggcgtgggtc caaggacgaa
      781 aagttaagat tcgccttcaa aatctacgac attgacaagg acggtttcat atccaatggt
      841 gagttgttca tcgtgttgaa gattatggta ggttctaatc tggacgatga acagctgcaa
      901 cagatagtag ataggacgat agtggaaaac gatagcgacg gcgacggacg tttaagtttc
      961 gaggagttta agaatgctat cgaaaccaca gaagtggcca agagtctgac attgcaatac
     1021 gatgtgtaag actaggggac acttcattca tttatggtat gccaatattt ttaagaaaag
     1081 aagaataata cgcgatattg ttttttaagg aaggaacgca cactcgccca gttagagtgc
     1141 tgatgatata tacatatata tatgtatatg taacaaacaa taagaagctt atcgataccg
     1201 tcgacctcga gggggggccc ggtaccagct tttgttccct ttagtgaggg ttaatttcga
     1261 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1321 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1381 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     1441 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     1501 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     1561 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     1621 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     1681 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     1741 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     1801 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     1861 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     1921 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     1981 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2041 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2101 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2161 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2221 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     2281 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     2341 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     2401 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     2461 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     2521 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     2581 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     2641 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     2701 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     2761 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     2821 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     2881 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     2941 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3001 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3061 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3121 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3181 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3241 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     3301 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     3361 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     3421 tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa attttttaat
     3481 ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg
     3541 ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc
     3601 tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca
     3661 cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat
     3721 ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa
     3781 cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta
     3841 aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat
     3901 cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtccta agaaaccatt
     3961 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     4021 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     4081 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4141 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatc
     4201 gactacgtcg ttaaggccgt ttctgacaga gtaaaattct tgagggaact ttcaccatta
     4261 tgggaaatgg ttcaagaagg tattgactta aactccatca aatggtcagg tcattgagtg
     4321 ttttttattt gttgtatttt ttttttttta gagaaaatcc tccaatatat aaattaggaa
     4381 tcatagtttc atgattttct gttacaccta actttttgtg tggtgccctc ctccttgtca
     4441 atattaatgt taaagtgcaa ttctttttcc ttatcacgtt gagccattag tatcaatttg
     4501 cttacctgta ttcctttaca tcctcctttt tctccttctt gataaatgta tgtagattgc
     4561 gtatatagtt tcgtctaccc tatgaacata ttccattttg taatttcgtg tcgtttctat
     4621 tatgaatttc atttataaag tttatgtaca aatatcataa aaaaagagaa tctttttaag
     4681 caaggatttt cttaacttct tcggcgacag catcaccgac ttcggtggta ctgttggaac
     4741 cacctaaatc accagttctg atacctgcat ccaaaacctt tttaactgca tcttcaatgg
     4801 ccttaccttc ttcaggcaag ttcaatgaca atttcaacat cattgcagca gacaagatag
     4861 tggcgatagg gttgacctta ttctttggca aatctggagc agaaccgtgg catggttcgt
     4921 acaaaccaaa tgcggtgttc ttgtctggca aagaggccaa ggacgcagat ggcaacaaac
     4981 ccaaggaacc tgggataacg gaggcttcat cggagatgat atcaccaaac atgttgctgg
     5041 tgattataat accatttagg tgggttgggt tcttaactag gatcatggcg gcagaatcaa
     5101 tcaattgatg ttgaaccttc aatgtaggga attcgttctt gatggtttcc tccacagttt
     5161 ttctccataa tcttgaagag gccaaaacat tagctttatc caaggaccaa ataggcaatg
     5221 gtggctcatg ttgtagggcc atgaaagcgg ccattcttgt gattctttgc acttctggaa
     5281 cggtgtattg ttcactatcc caagcgacac catcaccatc gtcttccttt ctcttaccaa
     5341 agtaaatacc tcccactaat tctctgacaa caacgaagtc agtaccttta gcaaattgtg
     5401 gcttgattgg agataagtct aaaagagagt cggatgcaaa gttacatggt cttaagttgg
     5461 cgtacaattg aagttcttta cggattttta gtaaaccttg ttcaggtcta acactaccgg
     5521 taccccattt aggaccaccc acagcaccta acaaaacggc atcagccttc ttggaggctt
     5581 ccagcgcctc atctggaagt ggaacacctg tagcatcgat agcagcacca ccaattaaat
     5641 gattttcgaa atcgaacttg acattggaac gaacatcaga aatagcttta agaaccttaa
     5701 tggcttcggc tgtgatttct tgaccaacgt ggtcacctgg caaaacgacg atcttcttag
     5761 gggcagacat tagaatggta tatccttgaa atatatatat atatattgct gaaatgtaaa
     5821 aggtaagaaa agttagaaag taagacgatt gctaaccacc tattggaaaa aacaataggt
     5881 ccttaaataa tattgtcaac ttcaagtatt gtgatgcaag catttagtca tgaacgcttc
     5941 tctattctat atgaaaagcc ggttccggcg ctctcacctt tcctttttct cccaattttt
     6001 cagttgaaaa aggtatatgc gtcaggcgac ctctgaaatt aacaaaaaat ttccagtcat
     6061 cgaatttgat tctgtgcgat agcgcccctg tgtgttctcg ttatgttgag gaaaaaaata
     6121 atggttgcta agagattcga actcttgcat cttacgatac ctgagtattc ccacagttaa
     6181 ctgcggtcaa gatatttctt gaatcaggcg ccttagaccg ctcggccaaa caaccaatta
     6241 cttgttgaga aatagagtat aattatccta taaatataac gtttttgaac acacatgaac
     6301 aaggaagtac aggacaattg attttgaaga gaatgtggat tttgatgtaa ttgttgggat
     6361 tccattttta ataaggcaat aatattaggt atgtagatat actagaagtt ctcctcgacc
     6421 ggtcgatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa
     6481 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     6541 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6601 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6661 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6721 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6781 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6841 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6901 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
     6961 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     7021 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgtt
//
