LOCUS       sequence-462097-        3687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for N-terminal tagging: PAC-3xTy-T2A-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3687)
  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 3687)
  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-NT-PTy
FEATURES             Location/Qualifiers
     source          1..3687
                     /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             1208..1261
                     /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(1466..1482)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1466..1482)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1479..1501)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1490..1506
                     /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(1514..1520,1521..1538,1539..1544))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1559..1580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1697..1714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1868..2456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1948..1967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2627..3418,3419..3487))
                     /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     3250..3269
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3488..3592)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3660..3678
                     /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 tatcgggccc gtataatgca gacctgctgc gaattcatga ctgaatacaa
      481 gccaacggtt cgtcttgcta cgcgagacga tgtgccgcgg gccgtacgga cgctggctgc
      541 tgcgttcgct gattatcccg ccacccgaca tacggttgac ccagatcgac acattgagcg
      601 agtcacagaa cttcaagagt tattcttaac acgggtgggc ttggacattg gtaaggtttg
      661 ggttgcggac gatggcgcgg ctgttgcggt gtggacgacg ccagagtcag tcgaagcggg
      721 cgcagtgttt gctgagatcg gtccccgaat ggcggagctt agcgggtcac gcctcgctgc
      781 acaacagcag atggaaggtc tgttggcacc acatagacct aaggagccgg cttggttctt
      841 agcgacagtt ggcgtcagtc cggaccacca aggtaagggg ctgggttcag cggttgttct
      901 tccaggagta gaggctgcgg aaagggcagg tgtgccggcg ttccttgaaa cgtctgcacc
      961 acgtaacctt cctttttacg agagattggg gtttacagta acagcagatg ttgaagtgcc
     1021 tgaagggccg aggacatggt gcatgacacg taagccggga gccccatggg aggtacacac
     1081 aaaccaggac ccactggacg gctccgaggt gcataccaat caagatccgc tggacggctc
     1141 tgaagttcac accaatcaag atcccttgga tggttctggt agtggttccg gttccggttc
     1201 tcgcgcggag ggtcgtggta gcctgctcac gtgtggtgac gtggaggaga acccaggtcc
     1261 gggatccgag gtacatacaa atcaggaccc cctcgatggt agtgaagtcc atacaaatca
     1321 ggacccgctg gatggctcag aggttcatac gaaccaagat cctcttgatg gatcaggatc
     1381 tggatcagga tcgggtagtg agctcctgca gggatgccgg gaccgacgag tgcagaggcg
     1441 tgcaagcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1501 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1561 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1621 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1681 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1741 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1801 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1861 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1921 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1981 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2041 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2101 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2161 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2221 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2281 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2341 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2401 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2461 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2521 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2581 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2641 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2701 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2761 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     2821 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2881 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2941 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3001 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3061 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3121 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3181 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3241 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3301 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3361 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3421 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3481 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3541 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3601 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     3661 ataggcgtat cacgaggccc tttcgtc
//
