LOCUS       sequence-363303-        7845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses HA-tagged 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 7845)
  TITLE     Downey Lab Plasmids
REFERENCE   2  (bases 1 to 7845)
  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-HA-PPK(mutant)
FEATURES             Location/Qualifiers
     source          1..7845
                     /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"
     CDS             3499..3525
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5596..5843
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5612..5630)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5861..5880)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5862..5880)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5890..5907)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5890..5906)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5899..5921)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6047..6502
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6134..6153)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6344..6365
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6633..7436)
                     /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(7437..7652)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7676..7695)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7795..7817
                     /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 agcttatgta cccatacgat gttccagatt acgctggtca ggaaaagcta
     3541 tacatcgaaa aagagctcag ttggttatcg ttcaatgaac gcgtgcttca ggaagcggcg
     3601 gacaaatcta acccgctgat tgaaaggatg cgtttcctgg ggatctattc caataacctt
     3661 gatgagttct ataaagtccg cttcgctgaa ctgaagcgac gcatcattat tagcgaagaa
     3721 caaggctcca actctcattc ccgccattta ctgggcaaaa ttcagtcccg ggtgctgaaa
     3781 gccgatcagg aattcgacgg cctctacaac gagctattgc tggagatggc gcgcaaccag
     3841 atcttcctga ttaatgaacg ccagctctcc gtcaatcaac aaaactggct gcgtcattat
     3901 tttaagcagt atctgcgtca gcacattacg ccgattttaa tcaatcctga cactgactta
     3961 gtgcagttcc tgaaagatga ttacacctat ctggcggtgg aaattatccg tggcgatacc
     4021 atccgttacg cgctgctgga gatcccatca gataaagtgc cgcgctttgt gaatttaccg
     4081 ccagaagcgc cgcgtcgacg caagccgatg attcttctgg ataacattct gcgttactgc
     4141 cttgatgata ttttcaaagg cttctttgat tatgacgcgc tgaatgccta ttcaatgaag
     4201 atgacccgcg atgccgaata cgatttagtg catgagatgg aagccagcct gatggagttg
     4261 atgtcttcca gtctcaagca gcgtttaact gctgagccgg tgcgttttgt ttatcagcgc
     4321 gatatgccca atgcgctggt tgaagtgtta cgcgaaaaac tgactatttc ccgctacgac
     4381 tccatcgtcc ccggcggtcg ttatcataat tttaaagact ttattaattt ccccaatgtc
     4441 ggcaaagcca atctggtgaa caaaccactg ccgcgtttac gccatatttg gtttgataaa
     4501 gcccagttcc gcaatggttt tgatgccatt cgcgaacgcg atgtgttgct ctattatcct
     4561 tatcacacct ttgagcatgt gctggaactg ctgcgtcagg cttcgttcga cccgagcgta
     4621 ctggcgatta aaattaacat ttaccgcgtg gcgaaagatt cacgcatcat cgactcgatg
     4681 atccacgccg cacataacgg taagaaagtc accgtggtgg ttgagttaca ggcgcgtttc
     4741 gacgaagaag ccaacattca ctgggcgaag cgcctgaccg aagcaggcgt gcacgttatc
     4801 ttctctgcgc cggggctgaa aattgcggcc aaactgttcc tgatttcacg taaagaaaac
     4861 ggtgaagtgg tgcgttatgc agcgatcggg accgggaact ttaacgaaaa aaccgcgcgt
     4921 ctttatactg actattcgtt gctgaccgcc gatgcgcgca tcaccaacga agtacggcgg
     4981 gtatttaact ttattgaaaa cccataccgt ccggtgacat ttgattattt aatggtatcg
     5041 ccgcaaaact cccgccgcct attgtatgaa atggtggacc gcgagatcgc caacgcgcag
     5101 caagggctgc ccagtggtat caccctgaag ctaaataacc ttgtcgataa aggcctggtt
     5161 gatcgtctgt atgcggcctc cagctccggc gtaccggtta atctgctggt tcgcggaatg
     5221 tgttcgctga tccccaatct ggaaggcatt agcgacaaca ttcgtgccat cagtattgtt
     5281 gaccgttacc ttgaagccga ccgggtttat atttttgaaa atggcggcga taaaaaggtc
     5341 tacctttctt ccgccgactg gatgacgcgc aatattgatt atcgtattga agtggcgacg
     5401 ccgctgctcg atccgcgcct gaagcagcgg gtactggaca tcatcgacat attgttcagc
     5461 gatacggtca aagcacgtta tatcgataaa gaactcagta atcgctacgt tccccgcggc
     5521 aatcgccgca aagtacgggc gcagttggcg atttatgact acatcaaatc actcgaacaa
     5581 cctgaataac tcgagtcatg taattagtta tgtcacgctt acattcacgc cctcccccca
     5641 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt
     5701 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc
     5761 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg
     5821 gacgctcgaa ggctttaatt tgcggccggt acccaattcg ccctatagtg agtcgtatta
     5881 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5941 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6001 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc
     6061 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6121 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     6181 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     6241 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     6301 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     6361 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     6421 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6481 aaaatattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6541 tttcacaccg catagggtaa taactgatat aattaaattg aagctctaat ttgtgagttt
     6601 agtatacatg catttactta taatacagtt ttttagtttt gctggccgca tcttctcaaa
     6661 tatgcttccc agcctgcttt tctgtaacgt tcaccctcta ccttagcatc ccttcccttt
     6721 gcaaatagtc ctcttccaac aataataatg tcagatcctg tagagaccac atcatccacg
     6781 gttctatact gttgacccaa tgcgtctccc ttgtcatcta aacccacacc gggtgtcata
     6841 atcaaccaat cgtaaccttc atctcttcca cccatgtctc tttgagcaat aaagccgata
     6901 acaaaatctt tgtcgctctt cgcaatgtca acagtaccct tagtatattc tccagtagat
     6961 agggagccct tgcatgacaa ttctgctaac atcaaaaggc ctctaggttc ctttgttact
     7021 tcttctgccg cctgcttcaa accgctaaca atacctgggc ccaccacacc gtgtgcattc
     7081 gtaatgtctg cccattctgc tattctgtat acacccgcag agtactgcaa tttgactgta
     7141 ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa aattgtactt ggcggataat
     7201 gcctttagcg gcttaactgt gccctccatg gaaaaatcag tcaagatatc cacatgtgtt
     7261 tttagtaaac aaattttggg acctaatgct tcaactaact ccagtaattc cttggtggta
     7321 cgaacatcca atgaagcaca caagtttgtt tgcttttcgt gcatgatatt aaatagcttg
     7381 gcagcaacag gactaggatg agtagcagca cgttccttat atgtagcttt cgacatgatt
     7441 tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg ttaagaatac tgggcaattt
     7501 catgtttctt caacactaca tatgcgtata tataccaatc taagtctgtg ctccttcctt
     7561 cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa
     7621 aaaaaagaat aaaaaaaaaa tgatgaattg aaaaggtggt atggtgcact ctcagtacaa
     7681 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     7741 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     7801 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcga
//
