LOCUS       sequence-338384-        7328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of L. major glcyoprotein-63 (D463N) with substrate 
            binding site mutation from chromosome 10 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7328)
  AUTHORS   Antonia AL, Barnes AB, Martin AT, Wang L, Ko DC
  TITLE     Variation in Leishmania chemokine suppression driven by 
            diversification of the GP63 virulence factor.
  JOURNAL   PLoS Negl Trop Dis. 2021 Oct 28;15(10):e0009224. doi: 
            10.1371/journal.pntd.0009224. eCollection 2021 Oct.
  PUBMED    34710089
REFERENCE   2  (bases 1 to 7328)
  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: "PLoS Negl 
            Trop Dis."; date: "2021-10-28"; volume: "15(10):e0009224. doi"; 
            pages: " 10.1371/journal.pntd.0009224. eCollection 2021 Oct"
COMMENT     Sequence Label: Lmajor_gp63_10_0460_D463N
FEATURES             Location/Qualifiers
     source          1..7328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        152..531
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        532..735
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     686..706
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     780..799
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        780..798
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2523..2552
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2553..2570
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2667..2684)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2673..2897
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2943..3371
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3030..3049)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3240..3261
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3380..3400)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3385..3714
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3565..3700
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3627..3646
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3763..4788
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3763..4788
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4918..5039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4955..4974)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5009..5028
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5088..5104)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5088..5104)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5101..5123)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5112..5128
                     /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(5136..5142,5143..5160,5161..5166))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5181..5202
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5319..5336)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5490..6078)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5570..5589)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6249..7040,7041..7109))
                     /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     6872..6891
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7110..7214)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7289..7308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
       61 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      121 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      241 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      301 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      361 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      421 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      481 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      541 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      601 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      661 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      721 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
      781 aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag cttggtaccg
      841 ccaccatgtg gtggaggctg tggtggctgc tgctgctgct gctgctgctg tggcctatgg
      901 tctgggctca cgccggggct ctgcagcaca gatgcgtgca cgatgcaatg caggcccggg
      961 tgagacagtc tgtggccgac caccacaagg cacctggagc cgtgagcgcc gtgggcctgc
     1021 catacgtgac actggatgca gcacacaccg cagcagcagc cgaccctcgg ccaggctctg
     1081 ccaggagcgt ggtgcgcgat gtgaactggg gcgccctgag aatcgccgtg agcacagagg
     1141 acctgaccga tcccgcatat cactgcgcca gggtgggaca gcacgtgaag gaccacgcag
     1201 gagccatcgt gacctgtaca gccgaggata tcctgaccaa tgagaagcgc gacatcctgg
     1261 tgaagcacct gatccctcag gccgtgcagc tgcacacaga gcggctgaag gtgcagcagg
     1321 tgcagggcaa gtggaaggtg accgacatgg tgggcgatat ctgcggcgac tttaaggtgc
     1381 cacaggccca catcacagag ggcttctcca acaccgattt tgtgatgtac gtggcctccg
     1441 tgccctctga ggagggcgtg ctggcatggg caaccacatg tcagaccttc tctgatggcc
     1501 acccagcagt gggcgtgatc aacatcccag cagcaaatat cgccagcagg tacgaccagc
     1561 tggtgacacg cgtggtgacc cacgagatgg cacacgcact gggcttttcc ggacccttct
     1621 ttaaggatgc caggatcgtg gcctctgtgc ccaacgtgcg cggcaagaat ttcgacgtgc
     1681 ctgtgatcaa cagctccaca gcagtggcaa aggcccgcga gcagtacgga tgcgataccc
     1741 tggagtatct ggaggtggag gaccagggag gagcaggcag cgccggctcc cacatcaaga
     1801 tgcggaatgc ccaggacgag ctgatggcac ctgcagcagc agcaggatac tatacagccc
     1861 tgaccatggc catcttccag gatctgggct tttaccaggc cgacttcagc aaggccgaag
     1921 tgatgccatg gggacagaac gcaggatgcg cctttctgac aaataagtgt atggagcaga
     1981 gcgtgaccca gtggcctgcc atgttctgca atgagtccga ggatgccatc cggtgtccaa
     2041 catctagact gagcctggga gcatgtggag tgaccagaca cccaggcctg ccaccttact
     2101 ggcagtactt caccgacccc agcctggcag gcctgagcgc cttcatggat tactgccccg
     2161 tggtggtgcc ttattccaac ggctcttgta cccagagggc atccgaggca cacgcctctc
     2221 tgctgccctt caacgtgttt tccgatgcag caaggtgcat cgacggagcc tttagaccta
     2281 aggccaccga tggcatcgtg aagtcttacg ccggcctgtg cgccaatgtg cagtgtgata
     2341 ccgccacaag aacctacagc gtgcaggtgc acggctccaa cgactataca aattgtaccc
     2401 caggcctgag ggtggagctg agcacagtgt ccaacgcctt cgagggagga ggatacatca
     2461 cctgcccacc ctatgtggag gtgtgccagg gcaatgtgca ggccgccaag gacggaggaa
     2521 acgaacagaa actgattagc gaagaagacc tgcaccacca ccaccaccat tgaggatcca
     2581 ctagtccagt gtggtggaat tctgcagata tccagcacag tggcggccgc tcgagtctag
     2641 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2701 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2761 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2821 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2881 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2941 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3001 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3061 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3121 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3181 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3241 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3301 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3361 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3421 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3481 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3541 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3601 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3661 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3721 ccgggagctt gtatatccat tttcggatct gatcagcacg tgatgaaaaa gcctgaactc
     3781 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     3841 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     3901 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     3961 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     4021 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     4081 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     4141 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     4201 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     4261 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     4321 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     4381 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     4441 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     4501 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     4561 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     4621 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     4681 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     4741 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtgctacga
     4801 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4861 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4921 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4981 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     5041 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     5101 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5161 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5221 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5281 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5341 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5401 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5461 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5521 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5581 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5641 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5701 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5761 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5821 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5881 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5941 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6001 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6061 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6121 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6181 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6241 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6301 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6361 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6421 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6481 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6541 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6601 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6661 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6721 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6781 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6841 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6901 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6961 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7021 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7081 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7141 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7201 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     7261 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7321 agtatctg
//
