LOCUS       sequence-462096-        4392 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-mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4392)
  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 4392)
  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-PmSc
FEATURES             Location/Qualifiers
     source          1..4392
                     /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(2171..2187)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2171..2187)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2184..2206)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2195..2211
                     /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(2219..2225,2226..2243,2244..2249))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2264..2285
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2402..2419)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2573..3161)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2653..2672)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3332..4123,4124..4192))
                     /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     3955..3974
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4193..4297)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4365..4383
                     /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 gggtaccgag gtacatacaa atcaggaccc cctcgatggt agtgaagtcc atacaaatca
     1321 ggacccgctg gatggctcag aggttcatac gaaccaagat cctcttgata agcttgtatc
     1381 aaagggcgaa gcagtaatca aggagttcat gcggtttaag gtacacatgg agggttccat
     1441 gaatggccat gaatttgaga tcgaaggaga gggggaaggg cgtccttacg aaggaacaca
     1501 aacagctaaa cttaaggtaa ctaagggggg acccctcccc ttttcctggg acatactgtc
     1561 accacaattc atgtacggca gccgagcctt tataaaacat ccagctgata tacccgatta
     1621 ttataagcaa agttttcccg agggctttaa gtgggaaagg gtcatgaact tcgaggacgg
     1681 gggtgcggta actgtaaccc aagacacctc tctcgaagac gggacgctta tttataaggt
     1741 gaaactccgg gggaccaatt ttccccctga cggacctgtc atgcagaaga agacaatggg
     1801 ctgggaggcg tccacagagc gcctgtatcc ggaggatggc gtacttaagg gtgacataaa
     1861 aatggcactt cgtttgaagg acggtggccg ttatctcgct gactttaaga caacttataa
     1921 ggcgaaaaag ccggtacaga tgccaggagc atacaatgtt gaccgcaaat tggacatcac
     1981 cagccacaac gaagactata ccgtcgttga acagtacgaa cggagtgaag ggaggcacag
     2041 tacaggcggg atggacgaat tgtataaagg atccggatca ggatctggat caggatcggg
     2101 tagtgagctc ctgcagggat gccgggaccg acgagtgcag aggcgtgcaa gcgagcttgg
     2161 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2221 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2281 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2341 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2401 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2461 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2521 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2581 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2641 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2701 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2761 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2821 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2881 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2941 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3001 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3061 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3121 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3181 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3241 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3301 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3361 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3421 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3481 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3541 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3601 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3661 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3721 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3781 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3841 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3901 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3961 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4021 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4081 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4141 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4201 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4261 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4321 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4381 ggccctttcg tc
//
