LOCUS       sequence-463623-        4395 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for C-terminal tagging: mScarlet-3xTy-T2A-PAC-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4395)
  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 4395)
  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-PmSc
FEATURES             Location/Qualifiers
     source          1..4395
                     /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             1320..1373
                     /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(2174..2190)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2174..2190)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2187..2209)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2198..2214
                     /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(2222..2228,2229..2246,2247..2252))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2267..2288
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2405..2422)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2576..3164)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2656..2675)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3335..4126,4127..4195))
                     /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     3958..3977
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4196..4300)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4368..4386
                     /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 aagcttgtat
      481 caaagggcga agcagtaatc aaggagttca tgcggtttaa ggtacacatg gagggttcca
      541 tgaatggcca tgaatttgag atcgaaggag agggggaagg gcgtccttac gaaggaacac
      601 aaacagctaa acttaaggta actaaggggg gacccctccc cttttcctgg gacatactgt
      661 caccacaatt catgtacggc agccgagcct ttataaaaca tccagctgat atacccgatt
      721 attataagca aagttttccc gagggcttta agtgggaaag ggtcatgaac ttcgaggacg
      781 ggggtgcggt aactgtaacc caagacacct ctctcgaaga cgggacgctt atttataagg
      841 tgaaactccg ggggaccaat tttccccctg acggacctgt catgcagaag aagacaatgg
      901 gctgggaggc gtccacagag cgcctgtatc cggaggatgg cgtacttaag ggtgacataa
      961 aaatggcact tcgtttgaag gacggtggcc gttatctcgc tgactttaag acaacttata
     1021 aggcgaaaaa gccggtacag atgccaggag catacaatgt tgaccgcaaa ttggacatca
     1081 ccagccacaa cgaagactat accgtcgttg aacagtacga acggagtgaa gggaggcaca
     1141 gtacaggcgg gatggacgaa ttgtataaag gatccgaggt acatacaaat caggaccccc
     1201 tcgatggtag tgaagtccat acaaatcagg acccgctgga tggctcagag gttcatacga
     1261 accaagatcc tcttgatgga tcaggatctg gatcaggatc gggtagtgag ctccgcgcgg
     1321 agggtcgtgg tagcctgctc acgtgtggtg acgtggagga gaacccaggt ccggctagca
     1381 tgactgaata caagccaacg gttcgtcttg ctacgcgaga cgatgtgccg cgggccgtac
     1441 ggacgctggc tgctgcgttc gctgattatc ccgccacccg acatacggtt gacccagatc
     1501 gacacattga gcgagtcaca gaacttcaag agttattctt aacacgggtg ggcttggaca
     1561 ttggtaaggt ttgggttgcg gacgatggcg cggctgttgc ggtgtggacg acgccagagt
     1621 cagtcgaagc gggcgcagtg tttgctgaga tcggtccccg aatggcggag cttagcgggt
     1681 cacgcctcgc tgcacaacag cagatggaag gtctgttggc accacataga cctaaggagc
     1741 cggcttggtt cttagcgaca gttggcgtca gtccggacca ccaaggtaag gggctgggtt
     1801 cagcggttgt tcttccagga gtagaggctg cggaaagggc aggtgtgccg gcgttccttg
     1861 aaacgtctgc accacgtaac cttccttttt acgagagatt ggggtttaca gtaacagcag
     1921 atgttgaagt gcctgaaggg ccgaggacat ggtgcatgac acgtaagccg ggagccctcg
     1981 aggaggtaca cacaaaccag gacccactgg acggctccga ggtgcatacc aatcaagatc
     2041 cgctggacgg ctctgaagtt cacaccaatc aagatccctt ggattaagca caggtctctc
     2101 aaattgggat gcatctgcag gatgccggga ccgacgagtg cagaggcgtg caagcgagct
     2161 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2221 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2281 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2341 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2401 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2461 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2521 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2581 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2641 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2701 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2761 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2821 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2881 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2941 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3001 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3061 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3121 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3181 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3241 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3301 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3361 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3421 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3481 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3541 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3601 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3661 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3721 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3781 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3841 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3901 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3961 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4021 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4081 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4141 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4201 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4261 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4321 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4381 cgaggccctt tcgtc
//
