LOCUS       sequence-338380-        7328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of L. major glcyoprotein-63 (P460G_D463N_S465A) with 
            substrate binding site mutations 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_P460G_D463N_S465A
FEATURES             Location/Qualifiers
     source          1..7328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        185..564
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        565..768
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     719..739
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     813..832
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        813..831
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2556..2585
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2586..2603
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2700..2717)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2706..2930
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2976..3404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3063..3082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3273..3294
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3413..3433)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3418..3747
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3598..3733
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3660..3679
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3796..4821
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3796..4821
                     /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    4951..5072
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4988..5007)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5042..5061
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5121..5137)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5121..5137)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5134..5156)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5145..5161
                     /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(5169..5175,5176..5193,5194..5199))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5214..5235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5352..5369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5523..6111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5603..5622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6282..7073,7074..7142))
                     /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     6905..6924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7143..7247)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(7322..7328,1..13))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
       61 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      121 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      181 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      241 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      301 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      361 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      421 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      481 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      541 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      601 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      661 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      721 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      781 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      841 gctggctagc gtttaaactt aagcttggta ccgccaccat gtggtggagg ctgtggtggc
      901 tgctgctgct gctgctgctg ctgtggccta tggtctgggc tcacgccggg gctctgcagc
      961 acagatgcgt gcacgatgca atgcaggccc gggtgagaca gtctgtggcc gaccaccaca
     1021 aggcacctgg agccgtgagc gccgtgggcc tgccatacgt gacactggat gcagcacaca
     1081 ccgcagcagc agccgaccct cggccaggct ctgccaggag cgtggtgcgc gatgtgaact
     1141 ggggcgccct gagaatcgcc gtgagcacag aggacctgac cgatcccgca tatcactgcg
     1201 ccagggtggg acagcacgtg aaggaccacg caggagccat cgtgacctgt acagccgagg
     1261 atatcctgac caatgagaag cgcgacatcc tggtgaagca cctgatccct caggccgtgc
     1321 agctgcacac agagcggctg aaggtgcagc aggtgcaggg caagtggaag gtgaccgaca
     1381 tggtgggcga tatctgcggc gactttaagg tgccacaggc ccacatcaca gagggcttct
     1441 ccaacaccga ttttgtgatg tacgtggcct ccgtgccctc tgaggagggc gtgctggcat
     1501 gggcaaccac atgtcagacc ttctctgatg gccacccagc agtgggcgtg atcaacatcc
     1561 cagcagcaaa tatcgccagc aggtacgacc agctggtgac acgcgtggtg acccacgaga
     1621 tggcacacgc actgggcttt tccggaccct tctttaagga tgccaggatc gtggcctctg
     1681 tgcccaacgt gcgcggcaag aatttcgacg tgcctgtgat caacagctcc acagcagtgg
     1741 caaaggcccg cgagcagtac ggatgcgata ccctggagta tctggaggtg gaggaccagg
     1801 gaggagcagg cagcgccggc tcccacatca agatgcggaa tgcccaggac gagctgatgg
     1861 cacctgcagc agcagcagga tactatacag ccctgaccat ggccatcttc caggatctgg
     1921 gcttttacca ggccgacttc agcaaggccg aagtgatgcc atggggacag aacgcaggat
     1981 gcgcctttct gacaaataag tgtatggagc agagcgtgac ccagtggcct gccatgttct
     2041 gcaatgagtc cgaggatgcc atccggtgtc caacatctag actgagcctg ggagcatgtg
     2101 gagtgaccag acacccaggc ctgccacctt actggcagta cttcaccgac cccagcctgg
     2161 caggcctgag cgccttcatg gattactgcc ccgtggtggt gggttattcc aacggcgctt
     2221 gtacccagag ggcatccgag gcacacgcct ctctgctgcc cttcaacgtg ttttccgatg
     2281 cagcaaggtg catcgacgga gcctttagac ctaaggccac cgatggcatc gtgaagtctt
     2341 acgccggcct gtgcgccaat gtgcagtgtg ataccgccac aagaacctac agcgtgcagg
     2401 tgcacggctc caacgactat acaaattgta ccccaggcct gagggtggag ctgagcacag
     2461 tgtccaacgc cttcgaggga ggaggataca tcacctgccc accctatgtg gaggtgtgcc
     2521 agggcaatgt gcaggccgcc aaggacggag gaaacgaaca gaaactgatt agcgaagaag
     2581 acctgcacca ccaccaccac cattgaggat ccactagtcc agtgtggtgg aattctgcag
     2641 atatccagca cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc
     2701 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2761 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2821 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2881 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     2941 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     3001 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3061 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3121 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3181 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3241 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3301 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3361 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg
     3421 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3481 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3541 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3601 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3661 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     3721 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     3781 tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     3841 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     3901 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     3961 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     4021 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     4081 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     4141 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     4201 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     4261 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     4321 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     4381 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     4441 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     4501 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     4561 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     4621 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     4681 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     4741 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     4801 cgtccgaggg caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat
     4861 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4921 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4981 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5041 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     5101 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     5161 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     5221 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5281 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5341 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5401 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5461 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5521 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5581 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5641 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5701 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5761 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5821 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5881 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5941 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     6001 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     6061 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     6121 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     6181 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     6241 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     6301 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6361 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6421 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6481 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6541 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6601 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6661 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6721 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6781 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6841 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6901 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6961 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     7021 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     7081 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     7141 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     7201 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     7261 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     7321 gtacaatc
//
