LOCUS       Exported                7963 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses mutated mouse PCSK3 in mammalian cells (histidine at 
            position 194 mutated to leucine via site directed mutagenesis).
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1-mPCSK3 H194L
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7963)
  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 7963)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7963
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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      2322..2331
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3461..3502
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3512..3529
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3552..3569)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3558..3782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3828..4256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3915..3934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4125..4146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4265..4285)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4270..4599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4450..4585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4512..4531
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4666..5460
                     /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(4720..4739)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5330..5349
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5636..5757
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5673..5692)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5727..5746
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5806..5822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5806..5822)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5819..5841)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5830..5846
                     /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(5854..5884)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5899..5920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6037..6054)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6208..6796)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6288..6307)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6967..7827)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGGRGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7590..7609
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7828..7932)
                     /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 taagcttggt accgagctcg gatccactag tccagcgtgg tggaattgcc cttagggcaa
      961 ttctgcagat atctgaccag gccagggaga caggcgctct ggggttccag tcacctgccc
     1021 ccccatggag ctgagatcct ggttgctatg ggtggtcgca gcagcaggag ccgtggtcct
     1081 gctggcagct gatgctcaag gccagaagat cttcaccaac acctgggccg tgcacattcc
     1141 tggaggccca gctgtggctg atagggtggc gcagaagcat ggcttccaca acctgggcca
     1201 gatcttcggt gactattacc acttctggca cagagcagtg acaaagcggt ccctgtcgcc
     1261 tcaccgcccg cggcacagcc ggctacagag ggagcctcaa gtaaagtggc tggagcagca
     1321 ggtagccaag cgaagagcca agagggacgt gtatcaggag cccacggacc ccaagttccc
     1381 ccagcagtgg tacctgtctg gtgtcactca gcgagacctg aatgtgaagg aggcctgggc
     1441 ccagggcttc acaggccatg gcattgtggt ctccatcctg gatgacggca ttgagaagaa
     1501 tcatcccgac ctagcaggca attatgaccc tggagccagt tttgacgtga atgaccagga
     1561 ccccgaccca cagcctcggt acacacagat gaatgacaac aggcttggca ctcgctgtgc
     1621 cggggaagtg gcagcagtgg ccaacaatgg tgtctgtggc gtaggtgtag cttacaatgc
     1681 ccgaattgga ggggtgcgga tgttggatgg cgaggtgact gatgcagtag aggcacgttc
     1741 gctgggcctg aatcccaacc acatccacat ctacagcgcc agctggggcc ctgaggacga
     1801 cggcaagacc gtggatggac cagcccggct cgctgaggag gccttctttc ggggagttag
     1861 ccagggccga ggagggctgg gctccatctt tgtctgggcc tcagggaatg ggggccggga
     1921 acatgacagc tgcaactgtg acggctacac caacagcatc tatacactgt ccatcagcag
     1981 cgccacacag ttcggcaatg tgccctggta cagtgaggcc tgctcctcca cactggccac
     2041 cacctacagc agcggcaacc agaatgagaa gcagatcgtg acaactgacc tgaggcagaa
     2101 gtgcacagaa tctcacacag gcacctcggc ttctgccccc ttggcagctg gtatcattgc
     2161 tctcaccctg gaggccaaca agaacctcac ctggcgggac atgcagcacc tggtagtgca
     2221 gacctcaaag ccagcccacc tcaacgctga tgattgggct accaacggcg tgggccggaa
     2281 agtgagccat tcgtatggct acgggctgtt ggatgccggt gccatggtgg ctctggccca
     2341 gaactggaca acagtggccc cccagcggaa gtgcattgtt gaaatcctgg tggaacccaa
     2401 ggacatcggc aaacggctag aggtgcgcaa ggcggtgaca gcatgcctgg gtgagcccaa
     2461 ccacatcacc aggctggaac acgttcaggc ccggctcacc ctgtcttaca atcgccgcgg
     2521 tgacctggct atccacctca tcagccccat gggcacccgc tccaccctgt tagctgccag
     2581 accacatgac tactctgctg atgggtttaa tgattgggct ttcatgacaa cccattcctg
     2641 ggatgaggat ccggctggcg aatgggtcct agagattgaa aataccagtg aagccaacaa
     2701 ctatgggacg ctgaccaagt tcactctcgt tctgtatggc acagcccccg aggggctctc
     2761 tacacctcca gaaagcagcg gctgcaagac cctcacatcc agccaggcct gcgtggtgtg
     2821 tgaggaaggt tactctctgc accagaaaag ctgtgtccag cactgcccac caggcttcat
     2881 cccccaagtc cttgatacac actacagcac tgagaatgat gtggagatca tccgtgccag
     2941 cgtctgtacc ccctgccacg cctcatgtgc cacttgccag ggcccggccc ccacagactg
     3001 cctcagctgc cccagccatg cctccctgga cccagtggag cagacctgct cccggcaaag
     3061 ccagagcagc cgggagtctc ggcctcagca gcagcctcct gcactgcgcc ctgaggtgga
     3121 gatggagccc cggctgcagg ctgggttggc ctctcacctg cccgaggtcc tggccggcct
     3181 cagctgcctt atcatcgtgc tcatctttgg catcgtcttc ctgttcctgc atcggtgttc
     3241 gggcttcagc ttccggggag tgaaagtgta caccatggac cgcggcctca tctcctacaa
     3301 ggggctgccc cctgaagcct ggcaggagga gtgcccatcg gactcagagg aggacgaggg
     3361 ccggggcgag aggaccgcct tcatcaaaga ccagagcgcc ctttgaaaag cccactgccc
     3421 accctatcaa atctcgagtc tagagggccc gcggttcgaa ggtaagccta tccctaaccc
     3481 tctcctcggt ctcgattcta cgcgtaccgg tcatcatcac catcaccatt gagtttaaac
     3541 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3601 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3661 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3721 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3781 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3841 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3901 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3961 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4021 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4081 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4141 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4201 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     4261 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4321 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4381 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4441 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4501 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4561 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4621 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4681 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4741 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4801 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4861 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4921 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4981 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5041 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5101 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     5161 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     5221 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5281 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5341 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5401 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5461 gcgggactct ggggttcgcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     5521 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5581 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5641 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5701 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5761 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5821 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5881 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5941 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6001 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     6061 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     6121 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     6181 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6241 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6301 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6361 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6421 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6481 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6541 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6601 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6661 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6721 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6781 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6841 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6901 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6961 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7021 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7081 tggccccagt gctgcaatga taccgcgaga cccacgccca ccggctccag atttatcagc
     7141 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7201 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7261 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7321 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     7381 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7441 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7501 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7561 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7621 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7681 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7741 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7801 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7861 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7921 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
