LOCUS       sequence-309851-        6785 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses N-terminally GFP-tagged and C-terminally Flag-tagged 
            EBA175 P1424A/Y1425I/Y1426I variant (residues 1284-1462) from 
            pcDNA3.1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6785)
  AUTHORS   Gandhi S, Baker RP, Cho S, Stanchev S, Strisovsky K, Urban S
  TITLE     Designed Parasite-Selective Rhomboid Inhibitors Block Invasion and 
            Clear Blood-Stage Malaria.
  JOURNAL   Cell Chem Biol. 2020 Aug 31. pii: S2451-9456(20)30333-0. doi: 
            10.1016/j.chembiol.2020.08.011.
  PUBMED    32888502
REFERENCE   2  (bases 1 to 6785)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Chem 
            Biol. 2020 Aug 31. pii: S2451-9456(20)30333-0. doi: 
            10.1016/j.chembiol.2020.08.011."
COMMENT     Sequence Label: pcDNA-GFP-EBA175-Flag(P1424I/Y1425I/Y1426I)
FEATURES             Location/Qualifiers
     source          1..6785
                     /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"
     CDS             join(1016..1018,1019..1021,1022..1732)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1016..1018,1019..1021,1022..1732)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1061..1082)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1322..1341)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1669..1690
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2306..2329
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2376..2393)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2382..2606
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2652..3080
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2739..2758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2949..2970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3089..3109)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3094..3423
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3274..3409
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3336..3355
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3490..4284
                     /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(3544..3563)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4154..4173
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4458..4579
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4495..4514)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4549..4568
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4628..4644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4628..4644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4641..4663)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4652..4668
                     /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(4676..4682,4683..4700,4701..4706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4721..4742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4859..4876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5030..5618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5110..5129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5789..6580,6581..6649))
                     /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     6412..6431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6650..6754)
                     /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 gctggctagc
      901 gtttaaactt aagcttacca tggtcccctc ggctggacag ctcgccctgt tcgctctggg
      961 tattgtgttg gctgcgtgcc aggccttgga gaacagcacg tccccgctgc gtacgatggt
     1021 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1081 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1141 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1201 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1261 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1321 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1381 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1441 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1501 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1561 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1621 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1681 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggccg
     1741 gactcaggga gcagaattct caggatccca tagccatcat ggtaatcgtc aggatcgtgg
     1801 tggtaatagc ggtaatgtgc tgaatatgcg cagcaacaat aacaacttca acaatatccc
     1861 gagccgctat aatctgtatg ataagaaact ggatctggat ctgtatgaaa atcgcaatga
     1921 tagcaccacc aaagaactga ttaaaaaact ggccgagatc aataagtgcg aaaatgaaat
     1981 tagcgtgaaa tattgcgatc acatgattca tgaagaaatt ccgctgaaaa cctgcaccaa
     2041 agaaaagacc cgtaatctgt gttgtgcagt gagcgattat tgcatgagct attttaccta
     2101 tgatagcgaa gaatattaca actgcaccaa acgcgaattt gatgatccga gctatacctg
     2161 ctttcgtaaa gaggcattta gcagcatgat cattattgcc ggtgccggtg ttctgttcat
     2221 cattctggtg attctgggtg caagccaggc aaaatatcag cgcctggaga aaatcaacaa
     2281 gaacaagatt gagaaaaacg tgaatgatta caaggacgat gacgataagt aggcggccgc
     2341 ataatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     2401 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     2461 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2521 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     2581 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     2641 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2701 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2761 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2821 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2881 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2941 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3001 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3061 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3121 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3181 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3241 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3301 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3361 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3421 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     3481 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     3541 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     3601 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     3661 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     3721 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     3781 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     3841 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     3901 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     3961 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     4021 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4081 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4141 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4201 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4261 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4321 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4381 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     4441 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4501 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4561 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     4621 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4681 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4741 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4801 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4861 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4921 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4981 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5041 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5101 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5161 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5221 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5281 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5341 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5401 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5461 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5521 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5581 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5641 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5701 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5761 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5821 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5881 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5941 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6001 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6061 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6781 acgtc
//
