LOCUS       sequence-313830-        7817 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses N-terminally GFP-tagged and C-terminally Flag-tagged AMA1 
            K546M/I547P/I548Y/I549Y variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7817)
  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 7817)
  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-AMA1-Flag(K546M/I547P/I548Y/I549Y)
FEATURES             Location/Qualifiers
     source          1..7817
                     /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             3338..3361
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3408..3425)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3414..3638
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3684..4112
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3771..3790)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3981..4002
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4121..4141)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4126..4455
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4306..4441
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4368..4387
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4522..5316
                     /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
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQHIAFYRLLDEFF"
     primer_bind     complement(4576..4595)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5186..5205
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5490..5611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5527..5546)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5581..5600
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5660..5676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5660..5676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5673..5695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5684..5700
                     /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(5708..5714,5715..5732,5733..5738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5753..5774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5891..5908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6062..6650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6142..6161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6821..7612,7613..7681))
                     /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     7444..7463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7682..7786)
                     /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 gcagaattca tcgaaatcgt ggaaagatcc aactacatgg gtaacccatg
     1801 gactgagtac atggcaaagt acgacatcga ggaagtgcac ggaagtggta tcagggttga
     1861 tctgggtgaa gatgccgaag tcgctggtac tcagtacaga ctcccttctg gtaagtgccc
     1921 tgttttcgga aagggtatca tcatcgaaaa ctctaagact actttcctca agcctgttgc
     1981 tactggtaac caagatctta aggacggagg tttcgctttc ccacctacta accctctgat
     2041 ctctccaatg actttgaacg gtatgcgtga cttctacaag aacaacgaat acgtcaagaa
     2101 cttggatgaa ttgactttgt gtagtagaca cgctggaaac atgaaccctg ataacgacaa
     2161 gaacagtaac tacaagtacc ccgcggttta cgactacaac gataagaagt gtcacatcct
     2221 gtacatcgct gcccaagaaa acaacggacc aagatactgt aacaaggatc aaagtaagag
     2281 aaactctatg ttctgtttca gacctgcaaa ggacaagctg ttcgaaaact acgtgtactt
     2341 gtccaagaac gttgtcgata actgggaaga agtctgccca agaaagaacc tcgagaacgc
     2401 aaagttcggt ctgtgggtcg atggtaactg tgaagacatc cctcatgtga acgagttcag
     2461 tgctaacgat ttgttcgagt gtaacaagct ggtcttcgag ttgtctgcca gtgaccaacc
     2521 taagcagtac gaacagcatt tgactgacta cgaaaagatc aaggaaggat tcaagaacaa
     2581 gaacgccgat atgatcaagt ccgctttcct cccaaccggt gcattcaaag cagatagata
     2641 caagtctcac ggtaagggtt acaactgggg aaactacaac agagaaaccc aaaagtgtga
     2701 aatcttcaac gtcaagccta cctgcctcat caacgacaag tcctacattg cgactactgc
     2761 cctgtctcat ccaatcgaag tcgaacacaa cttcccctgc agtctctaca aggacgagat
     2821 caagaaggaa atcgagcgtg aaagtaagcg tatcaagttg aacgataacg acgacgaagg
     2881 taacaagaag atcatcgcac ctaggatctt catctccgat gacaaggatt ccctcaagtg
     2941 tccttgtgac cctgagatgg tgagtcagtc cacttgtaga ttcttcgttt gcaagtgcgt
     3001 cgaacgtaga gccgaagtca ctagtaacaa cgaagttgtc gtgaaggaag aatacaagga
     3061 tgaatacgct gatattccag agcataagcc tacgtacgat aacatgatgc cgtattatgc
     3121 tagttctgct gctgtcgctg ttctggctac tatcctcatg gtgtaccttt acaagagaaa
     3181 gggaaacgct gagaagtacg acaagatgga tcaacctcaa cattacggta agagtacttc
     3241 caggaacgat gagatgttgg atccagaggc ctccttctgg ggtgaggaga agagagcctc
     3301 tcatactact ccagttttga tggagaagcc ttactacgat tacaaggacg atgacgataa
     3361 gtaggcggcc gcataatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3421 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3481 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3541 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3601 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3661 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3721 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3781 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3841 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3901 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3961 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4021 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4081 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4141 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4201 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4261 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4321 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4381 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4441 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4501 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4561 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4621 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4681 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4741 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4801 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4861 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4921 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4981 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5041 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5101 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5161 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5221 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcac
     5281 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5341 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5401 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5461 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5521 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5581 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5641 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5701 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5761 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5821 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5881 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5941 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6001 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6061 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6481 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6961 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     7741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     7801 aagtgccacc tgacgtc
//
