LOCUS       sequence-478567-        8364 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  SpAsp1 protein expression in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8364)
  AUTHORS   Raia P, Lee K, Bartsch SM, Rico-Resendiz F, Portugal-Calisto D, 
            Vadas O, Panse VG, Fiedler D, Hothorn M
  TITLE     A small signaling domain controls PPIP5K phosphatase activity in 
            phosphate homeostasis.
  JOURNAL   Nat Commun. 2025 Feb 19;16(1):1753. doi: 10.1038/s41467-025-56937-0.
  PUBMED    39966396
REFERENCE   2  (bases 1 to 8364)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-02-19"; volume: "16(1):1753. doi"; pages: " 
            10.1038/s41467-025-56937-0"
COMMENT     Sequence Label: pMH-SpAsp1
FEATURES             Location/Qualifiers
     source          1..8364
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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)."
     RBS             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3578..3598
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     CDS             3605..3628
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             3641..3928
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated and modified)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /note="modified to prevent cleavage by eukaryotic SUMO 
                     proteases"
                     /translation="DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRR
                     LMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             6713..6730
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6783..6801)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6797..6844
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6881..7336
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6968..6987)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7178..7199
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7428..8243)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7428..8243)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     8151..8170
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8235..8254
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgaaacat caccatcacc atcaccacgg
     3601 tgcttggagc cacccgcagt tcgaaaaggg atccctgcag gactcagaag tcaatcaaga
     3661 agctaagcca gaggtcaagc cagaagtcaa gcctgagact cacatcaatt taaaggtgtc
     3721 cgatggatct tcagagatct tcttcaagat caaaaagacc actcctttaa gaaggctgat
     3781 ggaagcgttc gctaaaagac agggtaagga aatggactcc ttaagattct tgtacgacgg
     3841 tattagaatt caagctgatc aggcccctga agatttggac atggaggata acgatattat
     3901 tgaggctcac cgcgaacaga ttggaggtgc catgatccag aacgcgtcac acctcaccag
     3961 cattgatact gaaagctcca cgcgaactgc aagtccagtc tcatctattg tgactccaac
     4021 caagcgtaac gttgtgggta tatgcgcgat ggacgcgaag gcgagatcga agccttgcag
     4081 aaacatcttg aatcgtataa tagctgaggg cgaattcgag gctattgtct ttggcgataa
     4141 tatgatactg gatgaggcag tagagaactg gccggcatgc gactacctga tctgctttta
     4201 tagctccggt ttccctctga agaaggccga gaaatacgtt gaactgagaa agccattctg
     4261 cgtgaatgac gttgtctttc aggaattact gtgggaccgg cgtctggtcc ttaatattct
     4321 cgacgcgatc agagtgtcta caccgcaaag actgatatgt tcccgcgacg gcgggccaaa
     4381 gatcaacaag gtgctcgagg agaaattacg ccgtaaattc ggtatcgaga ttaccgaagt
     4441 gcccacaccg gaagtcaaga tgctggatga ggacaccctg tcggtggacg ggaaaattat
     4501 caagaaacct tatgtcgaga agcctgttta cggcgaggac cataacatat acatctactt
     4561 tccgaagagt gtcggaggag gcggacgtaa gcttttcaga aaggtggcta acaaatcaag
     4621 tgactacgac ccggacttgt gcgcgccccg taccgagggt agcttcatat atgaagagtt
     4681 tatgaacgta gacaatgcag aggatgttaa agtgtacacg gttgggccgc actatagtca
     4741 cgccgagact cgtaagtctc cggttgtaga tggtatagtt cggcgaaacc cgcacggcaa
     4801 agagatccgc ttcattacta acctgtccga agaggagaag aacatggcta gtaaaatcag
     4861 tatagctttc gaacagcccg tctgcgggtt cgacttgctc cgggtgtctg gccaatctta
     4921 cgttatcgac gtcaacggtt ggagttttgt taaagacaat aatgactact acgacaacgc
     4981 ggcgcgcatc ttaaagcaga tgttccacgt ggcagagcgg caccgtagaa acagagtgcc
     5041 cagcgttcaa gaggtcttga acccgccacc acgtgagtct gaagcgtggc gtttgaagtc
     5101 tcttgtgggc gtattacgcc atgctgaccg gactccgaag cagaaattca aattctcgtt
     5161 caccagtgac cctttcgtca agctgctgca ggggcacaca gaagaggtga tactccggaa
     5221 tgagcagctg aactcagtcc tggcggcaac gaatctggca accgagctta agtgtgagga
     5281 cattaataaa ctgaagcaac tcagactcgc actggagacc aagaaggatc tccccggcac
     5341 taaggttcag ttaaagcctg catactctcc ggaaggtaaa ttgctgaagt tgcaattaat
     5401 aataaagtgg ggcggcgagt tcacccacag cgcacgctat cagagtaaag acctgggtga
     5461 acaattccac aaagatcttt acataatgaa ccgcgactgt ctcaaggacg ttgagatcta
     5521 taccagctcc gagcgtcggg tgtccgcctc ggcagagatc ttcgcgatgg cattcctgga
     5581 acaggagaca ataccgtcag atttgctgaa ggtgcgtaag gacttactgg acgactcgaa
     5641 cgccgcgaaa gacaccatgg acaaagtgaa gaagcatctt aagtcgcttc tccgggtggg
     5701 cgacacagca cgaaaagaat tcacatggcc tgagaatatg ccaaaaccct gcgaggtcat
     5761 gcagcaggtc gtccagttaa tgaagtacca ccgggcggtc atgcgcgaaa acttcataat
     5821 ccttggtccg gaagtagaac aggtgcagtc ccgatggtgc tgcaacgaga acccggcttt
     5881 attccgggag cgatgggaga aacttttctc agagttctgc gattcggaaa aggcggatcc
     5941 ctcgaaagtg agcgagcttt atgacactct gaaatacgac gccctgcata accgtcagtt
     6001 cctggagcgt attttcacgc cgtaccaata cttgaagctg ccgcaatctc cttctttaat
     6061 cgcgaaggaa ccgccgcagc ggacagactc gaacggaaac ttagtgggaa tgacgggagc
     6121 gaacaccaac catacagagc gcccattaga gaaactgtac gaactttatg acttggcgaa
     6181 ggtgctcttc gacttcgtct caccacagga gtacggcatc gagccaaagg agaaattgga
     6241 gataggcctg ttgacctctg tgcccttact gcgccagatt attcacgata tcaaggaagc
     6301 tcgtgactcg gaccacgcca gtacccgcat gtatttcacc aaggaatcac acatttacac
     6361 tctcctgaac tgcatcttag agagtggttt accgatgaag ctcccccgaa accagatccc
     6421 tgagctggac tacctgacac agatttgttt cgaattgttt gaacgtacca acccaagcgg
     6481 caacaaagag ttctcggtac ggataactct gtctccgggc tgctacgctc agtgccctct
     6541 cgacatgaac ctcgacgcaa agcattgcat atcagtctcc ccgcgtcgta gcctgacgcg
     6601 gcacctggat ctgcaacaat tcatcactaa gactgaagat ctgtgtaatt ctgtgcacct
     6661 gccaaagcga ttcatcccgg tcaatataaa ctgataagcg gccgcactcg agcaccacca
     6721 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     6781 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     6841 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     6901 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     6961 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     7021 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     7081 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7141 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7201 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     7261 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     7321 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     7381 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     7441 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     7501 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     7561 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     7621 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     7681 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     7741 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     7801 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     7861 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     7921 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     7981 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     8041 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     8101 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     8161 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     8221 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     8281 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     8341 cgtagaaaag atcaaaggat cttc
//
