LOCUS       sequence-462125-        3697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for C-terminal tagging: 3xTy-T2A-PAC-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3697)
  AUTHORS   Carbajo CG, Han X, Savur B, Upadhyaya A, Taha F, Tinti M, Wheeler 
            RJ, Yates PA, Tiengwe C
  TITLE     A high-throughput protein tagging toolkit that retains endogenous 
            untranslated regions for studying gene regulation in kinetoplastids.
  JOURNAL   Open Biol. 2025 Feb;15(2):240334. doi: 10.1098/rsob.240334. Epub 
            2025 Feb 26.
  PUBMED    39999874
REFERENCE   2  (bases 1 to 3697)
  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: "Open 
            Biol."; date: "2025-02"; volume: "15(2):240334. doi"; pages: " 
            10.1098/rsob.240334. Epub 2025 Feb 26"
COMMENT     Sequence Label: pRExT2A-CT-PTy
FEATURES             Location/Qualifiers
     source          1..3697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             621..674
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(1476..1492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1476..1492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1489..1511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1500..1516
                     /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(1524..1530,1531..1548,1549..1554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1569..1590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1707..1724)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1878..2466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1958..1977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2637..3428,3429..3497))
                     /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     3260..3279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3498..3602)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3670..3688
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa
      421 tgcgtcgaga tatcggtacc ggttctggta gtggttccgg ttccggttct aagcttgagg
      481 tacatacaaa tcaggacccc ctcgatggta gtgaagtcca tacaaatcag gacccgctgg
      541 atggctcaga ggttcatacg aaccaagatc ctcttgatgg atcaggatct ggatcaggat
      601 cgggtagtga gctccgcgcg gagggtcgtg gtagcctgct cacgtgtggt gacgtggagg
      661 agaacccagg tccggctagc atgactgaat acaagccaac ggttcgtctt gctacgcgag
      721 acgatgtgcc gcgggccgta cggacgctgg ctgctgcgtt cgctgattat cccgccaccc
      781 gacatacggt tgacccagat cgacacattg agcgagtcac agaacttcaa gagttattct
      841 taacacgggt gggcttggac attggtaagg tttgggttgc ggacgatggc gcggctgttg
      901 cggtgtggac gacgccagag tcagtcgaag cgggcgcagt gtttgctgag atcggtcccc
      961 gaatggcgga gcttagcggg tcacgcctcg ctgcacaaca gcagatggaa ggtctgttgg
     1021 caccacatag acctaaggag ccggcttggt tcttagcgac agttggcgtc agtccggacc
     1081 accaaggtaa ggggctgggt tcagcggttg ttcttccagg agtagaggct gcggaaaggg
     1141 caggtgtgcc ggcgttcctt gaaacgtctg caccacgtaa ccttcctttt tacgagagat
     1201 tggggtttac agtaacagca gatgttgaag tgcctgaagg gccgaggaca tggtgcatga
     1261 cacgtaagcc gggagccctc gaggaggtac acacaaacca ggacccactg gacggctccg
     1321 aggtgcatac caatcaagat ccgctggacg gctctgaagt tcacaccaat caagatccct
     1381 tggattaagc acaggtctct caaattggga tgcatctgca gggatgccgg gaccgacgag
     1441 tgcagaggcg tgcaagcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1501 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1561 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1621 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1681 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1741 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1801 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1861 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1921 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1981 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2041 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2101 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2161 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2221 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2281 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2341 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2401 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2461 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2521 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2581 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2641 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2701 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2761 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2821 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2881 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2941 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3001 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3061 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3121 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3181 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3241 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3301 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3361 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3421 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3481 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3541 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3601 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     3661 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
