LOCUS       sequence-431291-        7058 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone to express gene specific gRNA and carry the homology 
            repair template for CRISPR editing of Plasmodium berghei using the 
            PbHiT system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7058)
  AUTHORS   Jonsdottir TK, Paoletta MS, Ishizaki T, Hernandez S, Ivanova M, 
            Curbelo AH, Saiki PA, Selinger M, Das D, Henriksson J, Bushell ESC
  TITLE     A scalable CRISPR-Cas9 gene editing system facilitates CRISPR 
            screens in the malaria parasite Plasmodium berghei
  JOURNAL   bioRxiv 2024.04.20.590404
REFERENCE   2  (bases 1 to 7058)
  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: "bioRxiv 
            2024.04.20.590404"
COMMENT     Sequence Label: pPbHiT_3xmyc_hsp70UTR_hdhfr/yfcu
FEATURES             Location/Qualifiers
     source          1..7058
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             583..672
                     /codon_start=1
                     /product="3 tandem Myc epitope tags"
                     /label=3xMyc
                     /translation="EQKLISEEDLEQKLISEEDLEQKLISEEDL"
     promoter        1507..1611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1612..1680,1681..2472)
                     /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     complement(1830..1849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2643..3231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3132..3151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3385..3402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3519..3540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3555..3560,3561..3578,3579..3585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3593..3609
                     /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)."
     primer_bind     3598..3620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3617..3633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3617..3633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6911..6933)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tcgagaacta ttgtttcctt ttttgttttt ttatttttaa ttaaaatgta aatataaact
       61 cacaaaatga atgaataaat aaataaataa ataaataaaa aatgttaaat cgtgttaaac
      121 cgcgtgcttc atgtgatatg gcaagcgatt aatggccatt ttatccattt ttataatttg
      181 ataaaaaatt aaaacaaata aatgtaatgt taattatatt ttgtactata aatattattc
      241 tctacttcac gcaaatgtaa cactatccaa ctacactcat aatgtttaaa gaaaattggc
      301 attgttttac cattttttta aaacaaaaaa agaataaaat actttcgtat tatttagcta
      361 aaattgtact aaatttcata ccaaaaaaaa atatatatat gtatgcagct aatatttatt
      421 tgataagttt ttatgtaaat aatatttaat taaaaaaaaa attacatagc atgccgaatg
      481 cactattcat tttatggggg taattttttg agtaaccatt tttacttttt ttttacatat
      541 gcgcatactt cgagttatac aatattattg gagacgctgc aggaacaaaa acttattagc
      601 gaagaagatc ttgaacaaaa attaataagt gaagaagatt tagagcagaa gttgatttca
      661 gaggaggact tggtcgacgc ggccgcgtag ataaataatt ttttttttta tattattgtt
      721 ctgtacttct tttgtgaata gtatttttac atatgtactg ttaattttaa aatgtggaat
      781 gccacgaaca tattttttcg aacaaaatat atattttttg tgtcgaaata cataaatttg
      841 tgtacctcct ttttgttgta tatatgaaaa aggaataaag tgaatggagt cgaattactc
      901 tagaccctac aacttgtata tcactacttt ataatactat aatattatta ttaacattaa
      961 actatagaag aaatatcaac aatacttatg tatttgttca aactttatca gtatagtttg
     1021 gtaatcgcat atcatatatt tgttatgtgt aatattttat gaatatgcgt cttttttttt
     1081 aactataaat aattcaaaat aaaataacat aattaacatt tgcaagcata attaagatga
     1141 ttactttcac atttcatcca aagtggaaaa tgaccaccac atttgatcga agcttttacc
     1201 acttttgatg cattagctct gaaatgtata caagctttca tttgttaatg ttacaaaggg
     1261 aagtttttat ttgggggcac attaaaagtg aaaaaattaa aagttaaaaa agttaaaaaa
     1321 tgatcggtgt agttggtcat atagacatgt tgttatgcaa gtctttcaaa acgttcctca
     1381 taaaataaaa ggagggatct agagagcttt caattctcca aacggtatta ttggtatttt
     1441 ctgaaaactg cgtttccttg tttttattta tatttgacgt caggtggcac ttttcgggga
     1501 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1561 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1621 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1681 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1741 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1801 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     1861 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     1921 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     1981 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2041 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2101 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     2161 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2221 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2281 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2341 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2401 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2461 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2521 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2581 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2641 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2701 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2761 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     2821 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2881 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2941 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3001 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3061 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     3421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     3541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     3601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct tggtacccat
     3661 gccatggtcc ccgcggccgc tcgaggtgtt cgtttttctt atttatatat ttataccaat
     3721 tgattgtatt tataactgta aaaatgtgta tgttgtgtgc atattttttt ttgtgcatgc
     3781 acatgcatgt aaatagctaa aattatgaac attttatttt ttgttcagaa aaaaaaaact
     3841 ttacacacat aaaatggcta gtatgaatag ccatatttta tataaattaa atcctatgaa
     3901 tttatgacca tattaaaaat ttagatattt atggaacata atatgtttga aacaataaga
     3961 caaaattatt attattatta ttatttttac tgttataatt atgttgtctc ttcaatgatt
     4021 cataaatagt tggacttgat ttttaaaatg tttataatat gattagcata gttaaataaa
     4081 aaaagttgaa aaattaaaaa aaaacatata aacacaaatg atgttttttc cttcaatttc
     4141 gatatcatat agcaatcgta ttagccttaa gtttacaatt taatattcat actttaagta
     4201 ttttttgtag tatcctagat attgtgcttt aaatgctcac ccctcaaagc accagtaata
     4261 ttttcatcca ctgaaatacc attaaatttt caaaaaaata ctatgcatat aatgttatac
     4321 atataaacat aaaacgccat gtaaatcaaa aaatatataa aaatatgtat aaaaataaat
     4381 atgcactaaa tataagctaa ttatgcataa aaattaaagt gccctttatt aactagtcgt
     4441 aattatttat atttctatgt tataaaaaaa tcctcatata ataatataat taatatatgt
     4501 aatgtttttt ttattttata attttaatat aaaataatat gtaaattaat tcaaaaaata
     4561 aatataattg ttgtgaaaca aaaaacgtaa ttttttcatt tgccttcaaa atttaaattt
     4621 attttaatat ttcctaaaat atatatactt tgtgtataaa tatataaaaa tatatatttg
     4681 cttataaata aataaaaaat tttataaaac atagggggat ctatggttgg ttcgctaaac
     4741 tgcatcgtcg ctgtgtccca gaacatgggc atcggcaaga acggggacct gccctggcca
     4801 ccgctcagga acgaatttag atatttccag agaatgacca caacctcttc agtagaaggt
     4861 aaacagaatc tggtgattat gggtaagaag acctggttct ccattcctga gaagaatcga
     4921 cctttaaagg gtagaattaa tttagttctc agcagagaac tcaaggaacc tccacaagga
     4981 gctcattttc tttccagaag tctagatgat gccttaaaac ttactgaaca accagaatta
     5041 gcaaataaag tagacatggt ctggatagtt ggtggcagtt ctgtttataa ggaagccatg
     5101 aatcacccag gccatcttaa actatttgtg acaaggatca tgcaagactt tgaaagtgac
     5161 acgttttttc cagaaattga tttggagaaa tataaacttc tgccagaata cccaggtgtt
     5221 ctctctgatg tccaggagga gaaaggcatt aagtacaaat ttgaagtata tgagaagaat
     5281 gatgcaagcg gaggaggtgg atctggtgga ggtggaagtg caagcgtgac agggggaatg
     5341 gctagcaagt gggatcagaa gggtatggac attgcctatg aggaggcggc cttaggttac
     5401 aaggagggtg gtgttcctat tggcggatgt cttatcaata acaaagacgg aagtgttctc
     5461 ggtcgtggtc acaacatgag atttcaaaag ggatccgcca cactacatgg tgagatctcc
     5521 actttggaaa actgtgggag attagagggc aaagtgtaca aagataccac tttgtatacg
     5581 acgctgtctc catgcgacat gtgtacaggt gccatcatca tgtatggtat tccacgctgt
     5641 gttgtcggtg agaacgttaa tttcaaaagt aagggcgaga aatatttaca aactagaggt
     5701 cacgaggttg ttgttgttga cgatgagagg tgtaaaaaga tcatgaaaca atttatcgat
     5761 gaaagacctc aggattggtt tgaagatatt ggtgaggctt cggaaccatt taagaacgtc
     5821 tacttgctac ctcaaacaaa ccaattgctg ggtttgtaca ccatcatcag aaataagaat
     5881 acaactagac ctgatttcat tttctactcc gatagaatca tcagattgtt ggttgaagaa
     5941 ggtttgaacc atctacctgt gcaaaagcaa attgtggaaa ctgacaccaa cgaaaacttc
     6001 gaaggtgtct cattcatggg taaaatctgt ggtgtttcca ttgtcagagc tggtgaatcg
     6061 atggagcaag gattaagaga ctgttgtagg tctgtgcgta tcggtaaaat tttaattcaa
     6121 agggacgagg agactgcttt accaaagtta ttctacgaaa aattaccaga ggatatatct
     6181 gaaaggtatg tcttcctatt agacccaatg ctggccaccg gtggtagtgc tatcatggct
     6241 acagaagtct tgattaagag aggtgttaag ccagagagaa tttacttctt aaacctaatc
     6301 tgtagtaagg aagggattga aaaataccat gccgccttcc cagaggtcag aattgttact
     6361 ggtgccctcg acagaggtct agatgaaaac aagtatctag ttccagggtt gggtgacttt
     6421 ggtgacagat actactgtgt ttagggatcc gatcccgttt ttcttactta tatatttata
     6481 ccaattgatt gtatttataa ctgtaaaaat gtgtatgttg tgtgcatatt ttttttgtgc
     6541 atgcacatgc atgtaaatag ctaaaattat gaacatttta ttttttgttc agaaaaaaaa
     6601 aactttacac acataaaatg gctagtatga atagccatat tttatataaa ttaaatccta
     6661 tgaatttatg accatattaa aaatttagat atttatggaa cataatatgt ttgaaacaat
     6721 aagacaaaat tattattatt attattattt ttactgttat aattatgttg tctcttcaat
     6781 gattcataaa tagttggact tgatttttaa aatgtttata atatgattag catagttaaa
     6841 taaaaaaagt tgaaaaatta aaaaaaaaca tataaacaca aatgatgttt tttccttcaa
     6901 tttcggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     6961 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     7021 gcccttccca acagttgcgc agcctgaatg gcgaatga
//
