LOCUS       sequence-462093-        4408 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for C-terminal tagging: mNeongreen-3xTy-T2A-PAC-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4408)
  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 4408)
  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-PmNG
FEATURES             Location/Qualifiers
     source          1..4408
                     /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             1332..1385
                     /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(2187..2203)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2187..2203)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2200..2222)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2211..2227
                     /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(2235..2241,2242..2259,2260..2265))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2280..2301
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2418..2435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2589..3177)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2669..2688)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3348..4139,4140..4208))
                     /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     3971..3990
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4209..4313)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4381..4399
                     /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 aagcttgtct
      481 cgaaaggtga ggaagacaat atggccagct tgccagcaac gcacgagctt catatatttg
      541 ggtcgatcaa cggtgtggat ttcgacatgg tgggtcaagg cacaggtaat cctaatgacg
      601 ggtatgaaga actcaatttg aagagcacta agggagatct ccagttttcc ccgtggattc
      661 ttgtgccgca tattggttat ggttttcatc agtacttacc ataccccgat ggaatgtcgc
      721 ccttccaagc agctatggtg gacggctcgg ggtatcaagt acatcgcacc atgcaatttg
      781 aggacggagc atccctcacc gtaaactacc gctatacgta tgaggggagc cacataaaag
      841 gtgaggctca agttaaaggc acaggcttcc cagctgacgg cccggtcatg accaattcat
      901 taaccgctgc tgattggtgc cgttctaaga agacctaccc taacgacaag acgatcatta
      961 gtacttttaa gtggagttat accacaggaa acggaaagcg gtatcgaagc actgctcgaa
     1021 caacctacac atttgcgaag ccaatggccg cgaactactt gaagaaccag ccaatgtatg
     1081 tgtttcgaaa aacggaactg aagcattcga aaaccgagct taattttaaa gagtggcaga
     1141 aggcgtttac agacgtgatg gggatggacg aattgtataa gggatccgag gtacatacaa
     1201 atcaggaccc cctcgatggt agtgaagtcc atacaaatca ggacccgctg gatggctcag
     1261 aggttcatac gaaccaagat cctcttgatg gatcaggatc tggatcagga tcgggtagtg
     1321 agctccgcgc ggagggtcgt ggtagcctgc tcacgtgtgg tgacgtggag gagaacccag
     1381 gtccggctag catgactgaa tacaagccaa cggttcgtct tgctacgcga gacgatgtgc
     1441 cgcgggccgt acggacgctg gctgctgcgt tcgctgatta tcccgccacc cgacatacgg
     1501 ttgacccaga tcgacacatt gagcgagtca cagaacttca agagttattc ttaacacggg
     1561 tgggcttgga cattggtaag gtttgggttg cggacgatgg cgcggctgtt gcggtgtgga
     1621 cgacgccaga gtcagtcgaa gcgggcgcag tgtttgctga gatcggtccc cgaatggcgg
     1681 agcttagcgg gtcacgcctc gctgcacaac agcagatgga aggtctgttg gcaccacata
     1741 gacctaagga gccggcttgg ttcttagcga cagttggcgt cagtccggac caccaaggta
     1801 aggggctggg ttcagcggtt gttcttccag gagtagaggc tgcggaaagg gcaggtgtgc
     1861 cggcgttcct tgaaacgtct gcaccacgta accttccttt ttacgagaga ttggggttta
     1921 cagtaacagc agatgttgaa gtgcctgaag ggccgaggac atggtgcatg acacgtaagc
     1981 cgggagccct cgaggaggta cacacaaacc aggacccact ggacggctcc gaggtgcata
     2041 ccaatcaaga tccgctggac ggctctgaag ttcacaccaa tcaagatccc ttggattaag
     2101 cacaggtctc tcaaattggg atgcatctgc agggatgccg ggaccgacga gtgcagaggc
     2161 gtgcaagcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2221 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2281 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2341 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2401 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2461 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2521 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2581 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2641 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2701 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2761 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2821 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2881 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2941 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3001 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3061 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3121 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3181 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3241 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3301 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3361 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3421 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3481 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3541 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3601 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3661 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3721 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3781 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3841 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3901 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3961 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4021 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4081 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4141 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4201 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4261 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4321 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4381 aataggcgta tcacgaggcc ctttcgtc
//
