LOCUS       sequence-462130-        3480 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for N-terminal tagging: BSD-3xTy-T2A-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3480)
  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 3480)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-BTy
FEATURES             Location/Qualifiers
     source          1..3480
                     /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             1001..1054
                     /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(1259..1275)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1259..1275)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1272..1294)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1283..1299
                     /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(1307..1313,1314..1331,1332..1337))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1352..1373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1490..1507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1661..2249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1741..1760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2420..3211,3212..3280))
                     /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     3043..3062
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3281..3385)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3453..3471
                     /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 gaattcatgc ctttgtctca
      481 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
      541 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
      601 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
      661 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
      721 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
      781 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
      841 ttatgtgtgg gagggcccat gggaggtaca cacaaaccag gacccactgg acggctccga
      901 ggtgcatacc aatcaagatc cgctggacgg ctctgaagtt cacaccaatc aagatccctt
      961 ggatggttct ggtagtggtt ccggttccgg ttctcgcgcg gagggtcgtg gtagcctgct
     1021 cacgtgtggt gacgtggagg agaacccagg tccgggatcc gaggtacata caaatcagga
     1081 ccccctcgat ggtagtgaag tccatacaaa tcaggacccg ctggatggct cagaggttca
     1141 tacgaaccaa gatcctcttg atggatcagg atctggatca ggatcgggta gtgagctcct
     1201 gcagggatgc cgggaccgac gagtgcagag gcgtgcaagc gagcttggcg taatcatggt
     1261 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1321 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     1381 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     1441 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     1501 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1561 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     1621 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     1681 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     1741 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     1801 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     1861 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1921 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1981 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     2041 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     2101 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     2161 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     2221 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     2281 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     2341 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     2401 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     2461 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     2521 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     2581 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     2641 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     2701 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     2761 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     2821 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     2881 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     2941 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     3001 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     3061 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     3121 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     3181 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     3241 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     3301 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     3361 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     3421 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
