LOCUS       sequence-462116-        4201 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-BSD-3xTy.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4201)
  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 4201)
  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-BmNG
FEATURES             Location/Qualifiers
     source          1..4201
                     /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(1980..1996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1980..1996)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1993..2015)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2004..2020
                     /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(2028..2034,2035..2052,2053..2058))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2073..2094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2211..2228)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2382..2970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2462..2481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3141..3932,3933..4001))
                     /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     3764..3783
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4002..4106)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4174..4192
                     /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 catgcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     1441 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     1501 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     1561 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     1621 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     1681 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     1741 ggattcgtga attgctgccc tctggttatg tgtgggaggg cctcgaggag gtacacacaa
     1801 accaggaccc actggacggc tccgaggtgc ataccaatca agatccgctg gacggctctg
     1861 aagttcacac caatcaagat cccttggatt aagcacaggt ctctcaaatt gggatgcatc
     1921 tgcagggatg ccgggaccga cgagtgcaga ggcgtgcaag cgagcttggc gtaatcatgg
     1981 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2041 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2101 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2161 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2221 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2281 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2341 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2401 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2461 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     2521 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     2581 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     2641 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     2701 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     2761 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     2821 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     2881 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2941 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3001 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3061 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3121 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3181 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3241 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3301 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3361 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3421 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3481 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3541 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3601 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3661 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3721 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3781 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3841 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     3901 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     3961 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4021 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4081 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     4141 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     4201 c
//
