LOCUS       sequence-238767-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CEN LEU2 CNB1.
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: pTJK512
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(164..180)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(165..181)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(209..227)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(248..264)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(248..264)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(261..283)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    272..288
                     /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(296..302,303..320,321..326))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    341..362
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(479..496)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(650..1238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(730..749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1409..2200,2201..2269))
                     /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     2032..2051
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2270..2374)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2411..2914
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     2956..2974
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3012..3034)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3134..3153
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(3647..4741)
                     /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(4742..5149)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(5450..5905)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5587..5608)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5799..5818
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6031..6053
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6045..6062
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6046..6062
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6069..6088
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6069..6087
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     6120..6136
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6120..6136
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 gactagggga cacttcattc atttatggta tgccaatatt tttaagaaaa gaagaataat
       61 acgcgatatt gttttttaag gaaggaacgc acactcgccc agttagagtg ctgatgatat
      121 atacatatat atatgtatat gtaacaaaca ataagaagct tatcgatacc gtcgacctcg
      181 agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaatttcg agcttggcgt
      241 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
      301 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
      361 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
      421 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
      481 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
      541 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
      601 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
      661 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
      721 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
      781 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
      841 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
      901 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
      961 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1021 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1081 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1141 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1201 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1261 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1321 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1381 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1441 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1501 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     1561 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1621 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1681 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1741 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     1801 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     1861 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     1921 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     1981 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2041 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2101 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2161 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2221 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2281 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     2341 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     2401 ggtccttttc atcacgtgct ataaaaataa ttataattta aattttttaa tataaatata
     2461 taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa
     2521 gatgtaaaag actctagggg gatcgccaac aaatactacc ttttatcttg ctcttcctgc
     2581 tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat
     2641 cctgtgattt tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc
     2701 ttgtctaata aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt
     2761 attttttttt cttcattccg taactcttct accttcttta tttactttct aaaatccaaa
     2821 tacaaaacat aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag
     2881 atacgaggcg cgtgtaagtt acaggcaagc gatccgtcct aagaaaccat tattatcatg
     2941 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     3001 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3061 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3121 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat cgactacgtc
     3181 gttaaggccg tttctgacag agtaaaattc ttgagggaac tttcaccatt atgggaaatg
     3241 gttcaagaag gtattgactt aaactccatc aaatggtcag gtcattgagt gttttttatt
     3301 tgttgtattt tttttttttt agagaaaatc ctccaatata taaattagga atcatagttt
     3361 catgattttc tgttacacct aactttttgt gtggtgccct cctccttgtc aatattaatg
     3421 ttaaagtgca attctttttc cttatcacgt tgagccatta gtatcaattt gcttacctgt
     3481 attcctttac atcctccttt ttctccttct tgataaatgt atgtagattg cgtatatagt
     3541 ttcgtctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta ttatgaattt
     3601 catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa gcaaggattt
     3661 tcttaacttc ttcggcgaca gcatcaccga cttcggtggt actgttggaa ccacctaaat
     3721 caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg gccttacctt
     3781 cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata gtggcgatag
     3841 ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg tacaaaccaa
     3901 atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa cccaaggaac
     3961 ctgggataac ggaggcttca tcggagatga tatcaccaaa catgttgctg gtgattataa
     4021 taccatttag gtgggttggg ttcttaacta ggatcatggc ggcagaatca atcaattgat
     4081 gttgaacctt caatgtaggg aattcgttct tgatggtttc ctccacagtt tttctccata
     4141 atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat ggtggctcat
     4201 gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga acggtgtatt
     4261 gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca aagtaaatac
     4321 ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt ggcttgattg
     4381 gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg gcgtacaatt
     4441 gaagttcttt acggattttt agtaaacctt gttcaggtct aacactaccg gtaccccatt
     4501 taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct tccagcgcct
     4561 catctggaag tggaacacct gtagcatcga tagcagcacc accaattaaa tgattttcga
     4621 aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta atggcttcgg
     4681 ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta ggggcagaca
     4741 ttagaatggt atatccttga aatatatata tatatattgc tgaaatgtaa aaggtaagaa
     4801 aagttagaaa gtaagacgat tgctaaccac ctattggaaa aaacaatagg tccttaaata
     4861 atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc atgaacgctt ctctattcta
     4921 tatgaaaagc cggttccggc gctctcacct ttcctttttc tcccaatttt tcagttgaaa
     4981 aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa tttccagtca tcgaatttga
     5041 ttctgtgcga tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat aatggttgct
     5101 aagagattcg aactcttgca tcttacgata cctgagtatt cccacagtta actgcggtca
     5161 agatatttct tgaatcaggc gccttagacc gctcggccaa acaaccaatt acttgttgag
     5221 aaatagagta taattatcct ataaatataa cgtttttgaa cacacatgaa caaggaagta
     5281 caggacaatt gattttgaag agaatgtgga ttttgatgta attgttggga ttccattttt
     5341 aataaggcaa taatattagg tatgtagata tactagaagt tctcctcgac cggtcgatat
     5401 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     5461 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     5521 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     5581 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     5641 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     5701 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     5761 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     5821 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     5881 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     5941 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     6001 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     6061 gtgaattgta atacgactca ctatagggcg aattggagct ccaccgcggt ggcggccgct
     6121 ctagaactag tggatccacg ctatgaaaaa ggaaaacaaa tgactcttat aatttgtttc
     6181 cctcgacttc tctatttaaa tccaagattt taactaataa actatttaat aataaaacaa
     6241 ttaactcttt atatggaagc cttggaaatt agccgccaaa atgggatata cattccgtgc
     6301 gaagtgaccg cgtggaaggc cgggtatcat cttaaaaatc actagtttct tttttagcgg
     6361 aatgcaataa aggtgctttg tgctggtgtg gtttacacgg aacatctagt agctaaaact
     6421 tggtaactca atggtgatca gaatccatag aagcattttt atttcttaaa atgggtgctg
     6481 ctccttccaa aattgtggat ggtcttttag aagatacaaa ttgtatgtac acttcggagt
     6541 gaggaaaaga aagaaagggg aaattaaccg attttactaa cactgacact ttgaacagtt
     6601 gatagagatg aaattgaaag gttaaggaag agattcatga aattagatag agatagctca
     6661 gggtctattg ataaaaatga atttatgagc attcctggcg tttcgtcaaa ccctcttgct
     6721 ggacgtataa tggaggtttt cgatgctgat aatagtgggg acgtggattt tcaagagttc
     6781 atcacaggat tatccatttt cagtgggcgt gggtccaagg acgaaaagtt aagattcgcc
     6841 ttcaaaatct acgacattga caaggacggt ttcatatcca atggtgagtt gttcatcgtg
     6901 ttgaagatta tggtaggttc taatctggac gatgaacagc tgcaacagat agtagatagg
     6961 acgatagtgg aaaacgatag cgacggcgac ggacgtttaa gtttcgagga gtttaagaat
     7021 gctatcgaaa ccacagaagt ggccaagagt ctgacattgc aatacgatgt gtaa
//
