LOCUS       sequence-244746-        7823 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mutated mouse PCSK3 in mammalian cells (aspartic acid at 
            position 153 mutated to alanine via site directed mutagenesis).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7823)
  AUTHORS   Li JJ, Liu J, Lupino K, Liu X, Zhang L, Pei L
  TITLE     Growth Differentiation Factor 15 Maturation Requires Proteolytic 
            Cleavage by PCSK3, -5, and -6.
  JOURNAL   Mol Cell Biol. 2018 Oct 15;38(21). pii: MCB.00249-18. doi: 
            10.1128/MCB.00249-18. Print 2018 Nov 1.
  PUBMED    30104250
REFERENCE   2  (bases 1 to 7823)
  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"; journalName: "Mol Cell 
            Biol."; date: "2018-10-15"; volume: "38(21). pii: MCB.00249-18. 
            doi"; pages: " 10.1128/MCB.00249-18. Print 2018 Nov 1"
COMMENT     Sequence Label: pcDNA3.1-V5H6-mPCSK3 D153A
FEATURES             Location/Qualifiers
     source          1..7823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2209..2218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3321..3362
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3372..3389
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3412..3429)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3418..3642
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3688..4116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3775..3794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3985..4006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4125..4145)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4130..4459
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4310..4445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4526..5320
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4580..4599)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5190..5209
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5496..5617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5533..5552)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5587..5606
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5666..5682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5679..5701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5690..5706
                     /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(5714..5720,5721..5738,5739..5744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5759..5780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5897..5914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6068..6656)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6148..6167)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6827..7618,7619..7687))
                     /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     7450..7469
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7688..7792)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttcac catggagctg agatcctggt tgctatgggt ggtcgcagca gcaggagccg
      961 tggtcctgct ggcagctgat gctcaaggcc agaagatctt caccaacacc tgggccgtgc
     1021 acattcctgg aggcccagct gtggctgata gggtggcgca gaagcatggc ttccacaacc
     1081 tgggccagat cttcggtgac tattaccact tctggcacag agcagtgaca aagcggtccc
     1141 tgtcgcctca ccgcccgcgg cacagccggc tacagaggga gcctcaagta aagtggctgg
     1201 agcagcaggt agccaagcga agagccaaga gggacgtgta tcaggagccc acggacccca
     1261 agttccccca gcagtggtac ctgtctggtg tcactcagcg agacctgaat gtgaaggagg
     1321 cctgggccca gggcttcaca ggccatggca ttgtggtctc catcctggct gacggcattg
     1381 agaagaatca tcccgaccta gcaggcaatt atgaccctgg agccagtttt gacgtgaatg
     1441 accaggaccc cgacccacag cctcggtaca cacagatgaa tgacaacagg catggcactc
     1501 gctgtgccgg ggaagtggca gcagtggcca acaatggtgt ctgtggcgta ggtgtagctt
     1561 acaatgcccg aattggaggg gtgcggatgt tggatggcga ggtgactgat gcagtagagg
     1621 cacgttcgct gggcctgaat cccaaccaca tccacatcta cagcgccagc tggggccctg
     1681 aggacgacgg caagaccgtg gatggaccag cccggctcgc tgaggaggcc ttctttcggg
     1741 gagttagcca gggccgagga gggctgggct ccatctttgt ctgggcctca gggaatgggg
     1801 gccgggaaca tgacagctgc aactgtgacg gctacaccaa cagcatctat acactgtcca
     1861 tcagcagcgc cacacagttc ggcaatgtgc cctggtacag tgaggcctgc tcctccacac
     1921 tggccaccac ctacagcagc ggcaaccaga atgagaagca gatcgtgaca actgacctga
     1981 ggcagaagtg cacagaatct cacacaggca cctcggcttc tgcccccttg gcagctggta
     2041 tcattgctct caccctggag gccaacaaga acctcacctg gcgggacatg cagcacctgg
     2101 tagtgcagac ctcaaagcca gcccacctca acgctgatga ttgggctacc aacggcgtgg
     2161 gccggaaagt gagccattcg tatggctacg ggctgttgga tgccggtgcc atggtggctc
     2221 tggcccagaa ctggacaaca gtggcccccc agcggaagtg cattgttgaa atcctggtgg
     2281 aacccaagga catcggcaaa cggctagagg tgcgcaaggc ggtgacagca tgcctgggtg
     2341 agcccaacca catcaccagg ctggaacacg ttcaggcccg gctcaccctg tcttacaatc
     2401 gccgcggtga cctggctatc cacctcatca gccccatggg cacccgctcc accctgttag
     2461 ctgccagacc acatgactac tctgctgatg ggtttaatga ttgggctttc atgacaaccc
     2521 attcctggga tgaggatccg gctggcgaat gggtcctaga gattgaaaat accagtgaag
     2581 ccaacaacta tgggacgctg accaagttca ctctcgttct gtatggcaca gcccccgagg
     2641 ggctctctac acctccagaa agcagcggct gcaagaccct cacatccagc caggcctgcg
     2701 tggtgtgtga ggaaggttac tctctgcacc agaaaagctg tgtccagcac tgcccaccag
     2761 gcttcatccc ccaagtcctt gatacacact acagcactga gaatgatgtg gagatcatcc
     2821 gtgccagcgt ctgtaccccc tgccacgcct catgtgccac ttgccagggc ccggccccca
     2881 cagactgcct cagctgcccc agccatgcct ccctggaccc agtggagcag acctgctccc
     2941 ggcaaagcca gagcagccgg gagtctcggc ctcagcagca gcctcctgca ctgcgccctg
     3001 aggtggagat ggagccccgg ctgcaggctg ggttggcctc tcacctgccc gaggtcctgg
     3061 ccggcctcag ctgccttatc atcgtgctca tctttggcat cgtcttcctg ttcctgcatc
     3121 ggtgttcggg cttcagcttc cggggagtga aagtgtacac catggaccgc ggcctcatct
     3181 cctacaaggg gctgccccct gaagcctggc aggaggagtg cccatcggac tcagaggagg
     3241 acgagggccg gggcgagagg accgccttca tcaaagacca gagcgccctt cgctcgagtc
     3301 tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta
     3361 cgcgtaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     3421 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3481 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3541 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3601 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3661 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3721 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3781 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3841 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3901 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3961 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4021 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4081 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     4141 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4201 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4261 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4321 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4381 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4441 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4501 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4561 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4621 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4681 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4741 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4801 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4861 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4921 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4981 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5041 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5101 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5161 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5221 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5281 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgcg
     5341 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5401 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5461 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5521 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5581 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5641 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5701 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5941 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6001 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6061 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6121 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6181 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6241 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6301 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6361 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6421 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6481 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6541 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6601 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6661 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6721 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6781 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6841 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6901 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6961 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7021 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7081 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7141 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7201 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7261 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7321 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7381 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7441 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7501 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7561 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7621 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7681 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7741 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7801 cccgaaaagt gccacctgac gtc
//
