LOCUS       sequence-475916-        5989 bp    DNA     circular SYN 12-JUN-2025
DEFINITION  ScVip1-536-1107-K554A-K556A-K644A protein expression in insect 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5989)
  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 5989)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 12, 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: pBB3-ScVip1-536-1107-K554A-K556A-K644A
FEATURES             Location/Qualifiers
     source          1..5989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  779..944
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     polyA_signal    1065..1199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1089..1108)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1143..1162
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3110..3130)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(join(3146..3169,3170..3205,3206..3229))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(3239..3268)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     promoter        complement(3313..3404)
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     complement(3371..3388)
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     promoter        3449..3558
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             join(3600..3602,3603..3605,3606..4319)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3645..3666)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3906..3925)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4253..4274
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4364..4383)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4408..4456
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    4711..4744
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(4815..4820,4821..4837,4838..4843)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             5032..5565
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     mobile_element  5632..5855
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt tgacccaagt caaccggttg
      781 tgggcggaca aaatagttgg gaactgggag gggtggaaat ggagttttta aggattattt
      841 agggaagagt gacaaaatag atgggaactg ggtgtagcgt cgtaagctaa tacgaaaatt
      901 aaaaatgaca aaatagtttg gaactagatt tcacttatct ggttccaatt agatgggtat
      961 accctagggg ttatgatagt tattgctcag cggtggcagc agccaactca gcttcctttc
     1021 gggctttgtt agcagccgga tcttctaggc tcaagcagtg atcagatcca gacatgataa
     1081 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1141 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     1201 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     1261 aaagcaagta aaacctctac aaatgtggta tggctgatta tgatcctcta gtacttctcg
     1321 acaagcttgt cgagactgca ggctctagat tcgaaagtcc gattcgatcc tactcgagtg
     1381 cggccgctta tgaggtgatg ttcacgggag tgaactttgg aggcatgatc acacgggtga
     1441 acttgttacg cagcttctgc tgcacgtagt ccatgtcgag gtgcttggtc agggagatct
     1501 tagggatgca agagatgtag tgacggtcgt ccaattgcac gtccagaggg tcctgagtgt
     1561 ggcaaccggg agacatcttc agacggatag agtgggactt ctggccggaa gcgtcggtgg
     1621 actcgtacaa ctcgaaggtg atctgggaca agtagtccag ttcgggcaga gcgttgcgag
     1681 cgatcctcat ggggataccg ctttcgtaga tgatgttgag cagggtgtag atgtgggact
     1741 ctttggtgaa gtaagccaca caagcgggag tctcgcggtt cttcatgtcg ccgatgtcgt
     1801 tcaggatctg cttagccaga ggcagagaag tcagcaggcc gatgtccagc ttctcagcgt
     1861 cggagatacc gtactctttg gggcagatga agtcgaacag gaccttagcc agcttgtaca
     1921 gttcgcgcag ctgcatgaaa cggggctcgt cgaactggct agaggacttg gggttacgag
     1981 cggttgaggt ctttccagac tcgagcaccc agccgagaga acccacggag ttggaggagt
     2041 tcttgccgga gttgttcatg gagtgggagt cgatgatctt gaagttgttc ttggccagga
     2101 cgttgatggg gtaacgatcc accagggagt gagaaccgag ttcgtcagcg atagcctcgt
     2161 taggcaggcc ggggtcgaag atgttctcca agaactgacg gttgtgcaga gcgtcgtact
     2221 tcatggtgtc gtacagctcg gagatcttgg aagggtcgac cttctcggcg ttgttgaact
     2281 ctttgaaaag cttgtcccaa cgctctttga acaggctggg gtcctcagag gtgcaccaac
     2341 gggtctgcat ggagttcacg tctttcttgg cgaagttgtt atccatgatt ttcttgtggt
     2401 agttcatcag ctcgaccaca cgcttgatga ccaggtaagg ctcgggcatc ttggagggcc
     2461 aagcgaattg aggaggagcc tctttgccct cgcgcagcag gggcttcaac ttcttcttga
     2521 ctttgtccat gaggtcctta gcagcgttgg agtcgtccag caggtccttg cggatagaga
     2581 tctcgtcgga acccagctcg tcagcaccga acagagcacg ggtccagtac tgagcagtgt
     2641 gcagcacacg acgctcggag gagctgaaga tcttgatatt ctgcaggatg gacttgttca
     2701 acaggtcgaa gtcttgacgc atctgctctc ccagctcggt agcctggtac ttagcggagt
     2761 gagtaggctc gccaccccaa gccaggatga actgcacctt ctcgacctcg ttctctttgt
     2821 tcagcactgg cttcagctgg atcttggtgc cggggaagtt cagcttcttc tcgagagcgt
     2881 tagccagcac cttgatctta gcggggttgc cagccttctc gtccagagcg atacgcagag
     2941 cctgcagcac gatcttcaga tcgttcacgt tacggatgac gacttcctct ttgtgaccct
     3001 tcagcaggga gatgaagatg gggctagtga aggagtgctt gaaagcttga gcaggagtac
     3061 ggtcagcgtg acggatgatg atggcgaggc ccttgaacac ccatttggca ccttggaagt
     3121 acaggttctc gccggaaccg gagccctttt cgaattgagg gtggctccaa gcagaaccac
     3181 cggatccgcc accggaacca ccacccttct cgaactgggg gtgggaccag ctagcaccgt
     3241 ggtggtgatg gtgatgatga tgatgatgct tcatccgcgc ccgatggtgg gacggtatga
     3301 ataatccgga atatttatag gtttttttat tacaaaactg ttacgaaaac agtaaaatac
     3361 ttatttattt gcgagatggt tatcatttta attatctcca tgatctatta atattccgga
     3421 gtaggtcgcg aatcgatact agtatacgga cctttaattc aacccaacac aatatattat
     3481 agttaaataa gaattattat caaatcattt gtatattaat taaaatacta tactgtaaat
     3541 tacattttat ttacaatcac tcgacgaaga cttgatcacc cgggcggtcc gaaaccatga
     3601 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     3661 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     3721 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     3781 tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc
     3841 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     3901 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     3961 tgaaccgcat cgagctgaag ggtatcgact tcaaggagga cggcaacatc ctggggcaca
     4021 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     4081 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     4141 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     4201 acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     4261 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtagg
     4321 atcgaatcgg agcatgcggt accgggagat gggggaggct aactgaaaca cggaaggaga
     4381 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt
     4441 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     4501 ccaccgagac cccattggga ccaatacgcc cgcgtttctt ccttttcccc accccaaccc
     4561 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcaag ccctgccata
     4621 gccactacgg gtacgtttaa accttccgga gtaggtcgcg aatcgatact agtgtttaaa
     4681 ctcgctacct taggaccgtt atagttacag ataacttcgt ataatgtatg ctatacgaag
     4741 ttatggcgac ttcgctgctg cccaaggttg ccgggtgacg cacaccgtgg aaacggatga
     4801 aggcacgaac ccagttgaca taagcctgtt cggttcgtaa actgtaatgc aagtagcgta
     4861 tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg
     4921 cggttttcat ggcttgttat gactgttttt ttgtacagtc tatgcctcgg gcatccaagc
     4981 agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc
     5041 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttagg tggctcaagt
     5101 atgggcatca ttcgcacatg taggctcggc cctgaccaag tcaaatccat gcgggctgct
     5161 cttgatcttt tcggtcgtga gttcggagac gtagccacct actcccaaca tcagccggac
     5221 tccgattacc tcgggaactt gctccgtagt aagacattca tcgcgcttgc tgccttcgac
     5281 caagaagcgg ttgttggcgc tctcgcggct tacgttctgc ccaagtttga gcagccgcgt
     5341 agtgagatct atatctatga tctcgcagtc tccggcgagc accggaggca gggcattgcc
     5401 accgcgctca tcaatctcct caagcatgag gccaacgcgc ttggtgctta tgtgatctac
     5461 gtgcaagcag attacggtga cgatcccgca gtggctctct atacaaagtt gggcatacgg
     5521 gaagaagtga tgcactttga tatcgaccca agtaccgcca cctaacaatt cgttcaagcc
     5581 gagatcggct tcccggccgc ggagttgttc ggtaaattgt cacaacgccg cgaatatagt
     5641 ctttacatgc ccttggccac gcccctcttt aatacgacgg gcaatttgca cttcagaaaa
     5701 tgaagagttt gctttagcca taacaaaagt ccagtatgct ttttcacagc ataactggac
     5761 tgatttcagt ttacaactat tctgtctagt ttaagacttt attgtcatag tttagatcta
     5821 ttttgttcag tttaagactt tattgtccgc ccacacccgc ttacgcaggg catctgcgca
     5881 tccacaggaa gagcgaccca agtcaatcag gggataacgc aggaaagaac atgtgagcaa
     5941 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgtt
//
