LOCUS       sequence-363372-        7818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses catalytic mutant of E. coli PPK from yeast GPD promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7818)
  TITLE     Downey Lab Plasmids
REFERENCE   2  (bases 1 to 7818)
  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"
COMMENT     Sequence Label: p416-GPD-PPK(mutant)
FEATURES             Location/Qualifiers
     source          1..7818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2754..2774
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2799..3442
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3336..3359
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     3448..3464
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3448..3464
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      5569..5816
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5585..5603)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5834..5853)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5835..5853)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5863..5880)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5863..5879)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5872..5894)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6020..6475
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6107..6126)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6317..6338
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6606..7409)
                     /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(7410..7625)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7649..7668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7768..7790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
     2761 accctcacta aagggaacaa aagctggagc tcagtttatc attatcaata ctcgccattt
     2821 caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt
     2881 agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat
     2941 atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg
     3001 agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt
     3061 tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc
     3121 acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc tggagtaaat
     3181 gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta
     3241 ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa
     3301 attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt
     3361 aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta
     3421 aaacaccaga acttagtttc gacggattct agaactagtg gatcccccgg gctgcaggaa
     3481 ttcgatatca agcttatggg tcaggaaaag ctatacatcg aaaaagagct cagttggtta
     3541 tcgttcaatg aacgcgtgct tcaggaagcg gcggacaaat ctaacccgct gattgaaagg
     3601 atgcgtttcc tggggatcta ttccaataac cttgatgagt tctataaagt ccgcttcgct
     3661 gaactgaagc gacgcatcat tattagcgaa gaacaaggct ccaactctca ttcccgccat
     3721 ttactgggca aaattcagtc ccgggtgctg aaagccgatc aggaattcga cggcctctac
     3781 aacgagctat tgctggagat ggcgcgcaac cagatcttcc tgattaatga acgccagctc
     3841 tccgtcaatc aacaaaactg gctgcgtcat tattttaagc agtatctgcg tcagcacatt
     3901 acgccgattt taatcaatcc tgacactgac ttagtgcagt tcctgaaaga tgattacacc
     3961 tatctggcgg tggaaattat ccgtggcgat accatccgtt acgcgctgct ggagatccca
     4021 tcagataaag tgccgcgctt tgtgaattta ccgccagaag cgccgcgtcg acgcaagccg
     4081 atgattcttc tggataacat tctgcgttac tgccttgatg atattttcaa aggcttcttt
     4141 gattatgacg cgctgaatgc ctattcaatg aagatgaccc gcgatgccga atacgattta
     4201 gtgcatgaga tggaagccag cctgatggag ttgatgtctt ccagtctcaa gcagcgttta
     4261 actgctgagc cggtgcgttt tgtttatcag cgcgatatgc ccaatgcgct ggttgaagtg
     4321 ttacgcgaaa aactgactat ttcccgctac gactccatcg tccccggcgg tcgttatcat
     4381 aattttaaag actttattaa tttccccaat gtcggcaaag ccaatctggt gaacaaacca
     4441 ctgccgcgtt tacgccatat ttggtttgat aaagcccagt tccgcaatgg ttttgatgcc
     4501 attcgcgaac gcgatgtgtt gctctattat ccttatcaca cctttgagca tgtgctggaa
     4561 ctgctgcgtc aggcttcgtt cgacccgagc gtactggcga ttaaaattaa catttaccgc
     4621 gtggcgaaag attcacgcat catcgactcg atgatccacg ccgcacataa cggtaagaaa
     4681 gtcaccgtgg tggttgagtt acaggcgcgt ttcgacgaag aagccaacat tcactgggcg
     4741 aagcgcctga ccgaagcagg cgtgcacgtt atcttctctg cgccggggct gaaaattgcg
     4801 gccaaactgt tcctgatttc acgtaaagaa aacggtgaag tggtgcgtta tgcagcgatc
     4861 gggaccggga actttaacga aaaaaccgcg cgtctttata ctgactattc gttgctgacc
     4921 gccgatgcgc gcatcaccaa cgaagtacgg cgggtattta actttattga aaacccatac
     4981 cgtccggtga catttgatta tttaatggta tcgccgcaaa actcccgccg cctattgtat
     5041 gaaatggtgg accgcgagat cgccaacgcg cagcaagggc tgcccagtgg tatcaccctg
     5101 aagctaaata accttgtcga taaaggcctg gttgatcgtc tgtatgcggc ctccagctcc
     5161 ggcgtaccgg ttaatctgct ggttcgcgga atgtgttcgc tgatccccaa tctggaaggc
     5221 attagcgaca acattcgtgc catcagtatt gttgaccgtt accttgaagc cgaccgggtt
     5281 tatatttttg aaaatggcgg cgataaaaag gtctaccttt cttccgccga ctggatgacg
     5341 cgcaatattg attatcgtat tgaagtggcg acgccgctgc tcgatccgcg cctgaagcag
     5401 cgggtactgg acatcatcga catattgttc agcgatacgg tcaaagcacg ttatatcgat
     5461 aaagaactca gtaatcgcta cgttccccgc ggcaatcgcc gcaaagtacg ggcgcagttg
     5521 gcgatttatg actacatcaa atcactcgaa caacctgaat aactcgagtc atgtaattag
     5581 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     5641 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     5701 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     5761 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcggcc
     5821 ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     5881 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     5941 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6001 cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     6061 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     6121 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     6181 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     6241 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     6301 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     6361 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     6421 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttcctga
     6481 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataggg taataactga
     6541 tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac ttataataca
     6601 gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc ttttctgtaa
     6661 cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc aacaataata
     6721 atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc caatgcgtct
     6781 cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc ttcatctctt
     6841 ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct cttcgcaatg
     6901 tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga caattctgct
     6961 aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt caaaccgcta
     7021 acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc tgctattctg
     7081 tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa ttttctgtct
     7141 tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac tgtgccctcc
     7201 atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt gggacctaat
     7261 gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc acacaagttt
     7321 gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg atgagtagca
     7381 gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca ggtttttgtt
     7441 ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact acatatgcgt
     7501 atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc ggagattacc
     7561 gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag aataaaaaaa aaatgatgaa
     7621 ttgaaaaggt ggtatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7681 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7741 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7801 catcaccgaa acgcgcga
//
