LOCUS       sequence-462123-        4189 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-BSD-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4189)
  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 4189)
  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-BmSc
FEATURES             Location/Qualifiers
     source          1..4189
                     /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(1968..1984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1968..1984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1981..2003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1992..2008
                     /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(2016..2022,2023..2040,2041..2046))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2061..2082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2199..2216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2370..2958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2450..2469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3129..3920,3921..3989))
                     /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     3752..3771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3990..4094)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4162..4180
                     /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 tgcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca
     1441 tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct
     1501 tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg
     1561 gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga
     1621 acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg
     1681 ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat
     1741 tgctgccctc tggttatgtg tgggagggcc tcgaggaggt acacacaaac caggacccac
     1801 tggacggctc cgaggtgcat accaatcaag atccgctgga cggctctgaa gttcacacca
     1861 atcaagatcc cttggattaa gcacaggtct ctcaaattgg gatgcatctg cagggatgcc
     1921 gggaccgacg agtgcagagg cgtgcaagcg agcttggcgt aatcatggtc atagctgttt
     1981 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     2041 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     2101 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2161 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     2221 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     2281 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     2341 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2401 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2461 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2521 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2581 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2641 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2701 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2761 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2821 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2881 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2941 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3001 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3061 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3121 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3181 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3241 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3301 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3361 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3421 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3481 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3541 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3601 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3661 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3721 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3781 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3841 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3901 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3961 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4021 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4081 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4141 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
