LOCUS       sequence-238936-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CEN LEU2 CNB1mutEF4.
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: pTJK516
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1119..1135)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1120..1136)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(1164..1182)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1203..1219)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1203..1219)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1216..1238)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1227..1243
                     /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(1251..1257,1258..1275,1276..1281))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1296..1317
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1434..1451)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1605..2193)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1685..1704)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2364..3155,3156..3224))
                     /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     2987..3006
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3225..3329)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    3366..3869
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3911..3929
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3967..3989)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4089..4108
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(4602..5696)
                     /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(5697..6104)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(6405..6860)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6542..6563)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6754..6773
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6986..7008
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7000..7017
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7001..7017
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7024..7043
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7024..7042
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tctagaacta gtggatccac gctatgaaaa aggaaaacaa atgactctta taatttgttt
       61 ccctcgactt ctctatttaa atccaagatt ttaactaata aactatttaa taataaaaca
      121 attaactctt tatatggaag ccttggaaat tagccgccaa aatgggatat acattccgtg
      181 cgaagtgacc gcgtggaagg ccgggtatca tcttaaaaat cactagtttc ttttttagcg
      241 gaatgcaata aaggtgcttt gtgctggtgt ggtttacacg gaacatctag tagctaaaac
      301 ttggtaactc aatggtgatc agaatccata gaagcatttt tatttcttaa aatgggtgct
      361 gctccttcca aaattgtgga tggtctttta gaagatacaa attgtatgta cacttcggag
      421 tgaggaaaag aaagaaaggg gaaattaacc gattttacta acactgacac tttgaacagt
      481 tgatagagat gaaattgaaa ggttaaggaa gagattcatg aaattagata gagatagctc
      541 agggtctatt gataaaaatg aatttatgag cattcctggc gtttcgtcaa accctcttgc
      601 tggacgtata atggaggttt tcgatgctga taatagtggg gacgtggatt ttcaagagtt
      661 catcacagga ttatccattt tcagtgggcg tgggtccaag gacgaaaagt taagattcgc
      721 cttcaaaatc tacgacattg acaaggacgg tttcatatcc aatggtgagt tgttcatcgt
      781 gttgaagatt atggtaggtt ctaatctgga cgatgaacag ctgcaacaga tagtagatag
      841 gacgatagtg gaaaacgcta gcgacggcga cggacgttta agtttcgagg agtttaagaa
      901 tgctatcgaa accacagaag tggccaagag tctgacattg caatacgatg tgtaagacta
      961 ggggacactt cattcattta tggtatgcca atatttttaa gaaaagaaga ataatacgcg
     1021 atattgtttt ttaaggaagg aacgcacact cgcccagtta gagtgctgat gatatataca
     1081 tatatatatg tatatgtaac aaacaataag aagcttatcg ataccgtcga cctcgagggg
     1141 gggcccggta ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca
     1201 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1261 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1441 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1501 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1561 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1621 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1681 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1741 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     1801 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     1861 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     1921 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     1981 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2041 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2101 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2161 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2221 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2281 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2341 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2401 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2461 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2521 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2581 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2641 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2701 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     2761 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     2821 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     2881 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     2941 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3001 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3061 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3121 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3181 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3241 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3301 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc
     3361 ttttcatcac gtgctataaa aataattata atttaaattt tttaatataa atatataaat
     3421 taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt
     3481 aaaagactct agggggatcg ccaacaaata ctacctttta tcttgctctt cctgctctca
     3541 ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag accacacacg aaaatcctgt
     3601 gattttacat tttacttatc gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc
     3661 taataaatat atatgtaaag tacgcttttt gttgaaattt tttaaacctt tgtttatttt
     3721 tttttcttca ttccgtaact cttctacctt ctttatttac tttctaaaat ccaaatacaa
     3781 aacataaaaa taaataaaca cagagtaaat tcccaaatta ttccatcatt aaaagatacg
     3841 aggcgcgtgt aagttacagg caagcgatcc gtcctaagaa accattatta tcatgacatt
     3901 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     3961 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4021 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     4081 taactatgcg gcatcagagc agattgtact gagagtgcac catatcgact acgtcgttaa
     4141 ggccgtttct gacagagtaa aattcttgag ggaactttca ccattatggg aaatggttca
     4201 agaaggtatt gacttaaact ccatcaaatg gtcaggtcat tgagtgtttt ttatttgttg
     4261 tatttttttt tttttagaga aaatcctcca atatataaat taggaatcat agtttcatga
     4321 ttttctgtta cacctaactt tttgtgtggt gccctcctcc ttgtcaatat taatgttaaa
     4381 gtgcaattct ttttccttat cacgttgagc cattagtatc aatttgctta cctgtattcc
     4441 tttacatcct cctttttctc cttcttgata aatgtatgta gattgcgtat atagtttcgt
     4501 ctaccctatg aacatattcc attttgtaat ttcgtgtcgt ttctattatg aatttcattt
     4561 ataaagttta tgtacaaata tcataaaaaa agagaatctt tttaagcaag gattttctta
     4621 acttcttcgg cgacagcatc accgacttcg gtggtactgt tggaaccacc taaatcacca
     4681 gttctgatac ctgcatccaa aaccttttta actgcatctt caatggcctt accttcttca
     4741 ggcaagttca atgacaattt caacatcatt gcagcagaca agatagtggc gatagggttg
     4801 accttattct ttggcaaatc tggagcagaa ccgtggcatg gttcgtacaa accaaatgcg
     4861 gtgttcttgt ctggcaaaga ggccaaggac gcagatggca acaaacccaa ggaacctggg
     4921 ataacggagg cttcatcgga gatgatatca ccaaacatgt tgctggtgat tataatacca
     4981 tttaggtggg ttgggttctt aactaggatc atggcggcag aatcaatcaa ttgatgttga
     5041 accttcaatg tagggaattc gttcttgatg gtttcctcca cagtttttct ccataatctt
     5101 gaagaggcca aaacattagc tttatccaag gaccaaatag gcaatggtgg ctcatgttgt
     5161 agggccatga aagcggccat tcttgtgatt ctttgcactt ctggaacggt gtattgttca
     5221 ctatcccaag cgacaccatc accatcgtct tcctttctct taccaaagta aatacctccc
     5281 actaattctc tgacaacaac gaagtcagta cctttagcaa attgtggctt gattggagat
     5341 aagtctaaaa gagagtcgga tgcaaagtta catggtctta agttggcgta caattgaagt
     5401 tctttacgga tttttagtaa accttgttca ggtctaacac taccggtacc ccatttagga
     5461 ccacccacag cacctaacaa aacggcatca gccttcttgg aggcttccag cgcctcatct
     5521 ggaagtggaa cacctgtagc atcgatagca gcaccaccaa ttaaatgatt ttcgaaatcg
     5581 aacttgacat tggaacgaac atcagaaata gctttaagaa ccttaatggc ttcggctgtg
     5641 atttcttgac caacgtggtc acctggcaaa acgacgatct tcttaggggc agacattaga
     5701 atggtatatc cttgaaatat atatatatat attgctgaaa tgtaaaaggt aagaaaagtt
     5761 agaaagtaag acgattgcta accacctatt ggaaaaaaca ataggtcctt aaataatatt
     5821 gtcaacttca agtattgtga tgcaagcatt tagtcatgaa cgcttctcta ttctatatga
     5881 aaagccggtt ccggcgctct cacctttcct ttttctccca atttttcagt tgaaaaaggt
     5941 atatgcgtca ggcgacctct gaaattaaca aaaaatttcc agtcatcgaa tttgattctg
     6001 tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa aaaataatgg ttgctaagag
     6061 attcgaactc ttgcatctta cgatacctga gtattcccac agttaactgc ggtcaagata
     6121 tttcttgaat caggcgcctt agaccgctcg gccaaacaac caattacttg ttgagaaata
     6181 gagtataatt atcctataaa tataacgttt ttgaacacac atgaacaagg aagtacagga
     6241 caattgattt tgaagagaat gtggattttg atgtaattgt tgggattcca tttttaataa
     6301 ggcaataata ttaggtatgt agatatacta gaagttctcc tcgaccggtc gatatgcggt
     6361 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa
     6421 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     6481 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     6541 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     6601 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     6661 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     6721 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     6781 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6841 tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     6901 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     6961 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
     7021 ttgtaatacg actcactata gggcgaattg gagctccacc gcggtggcgg ccgc
//
