LOCUS       sequence-463546-        4196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for N-terminal tagging: BSD-3xTy-T2A-3xTy-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4196)
  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 4196)
  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-NT-BmNG
FEATURES             Location/Qualifiers
     source          1..4196
                     /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(1975..1991)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1975..1991)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1988..2010)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1999..2015
                     /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(2023..2029,2030..2047,2048..2053))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2068..2089
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2206..2223)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2377..2965)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2457..2476)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3136..3927,3928..3996))
                     /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     3759..3778
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3997..4101)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4169..4187
                     /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 tccgggtacc gaggtacata caaatcagga
     1081 ccccctcgat ggtagtgaag tccatacaaa tcaggacccg ctggatggct cagaggttca
     1141 tacgaaccaa gatcctcttg ataagcttgt ctcgaaaggt gaggaagaca atatggccag
     1201 cttgccagca acgcacgagc ttcatatatt tgggtcgatc aacggtgtgg atttcgacat
     1261 ggtgggtcaa ggcacaggta atcctaatga cgggtatgaa gaactcaatt tgaagagcac
     1321 taagggagat ctccagtttt ccccgtggat tcttgtgccg catattggtt atggttttca
     1381 tcagtactta ccataccccg atggaatgtc gcccttccaa gcagctatgg tggacggctc
     1441 ggggtatcaa gtacatcgca ccatgcaatt tgaggacgga gcatccctca ccgtaaacta
     1501 ccgctatacg tatgagggga gccacataaa aggtgaggct caagttaaag gcacaggctt
     1561 cccagctgac ggcccggtca tgaccaattc attaaccgct gctgattggt gccgttctaa
     1621 gaagacctac cctaacgaca agacgatcat tagtactttt aagtggagtt ataccacagg
     1681 aaacggaaag cggtatcgaa gcactgctcg aacaacctac acatttgcga agccaatggc
     1741 cgcgaactac ttgaagaacc agccaatgta tgtgtttcga aaaacggaac tgaagcattc
     1801 gaaaaccgag cttaatttta aagagtggca gaaggcgttt acagacgtga tggggatgga
     1861 cgaattgtat aagggatccg gatcaggatc tggatcagga tcgggtagtg agctcctgca
     1921 ggatgccggg accgacgagt gcagaggcgt gcaagcgagc ttggcgtaat catggtcata
     1981 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2041 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2101 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2161 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2221 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2281 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2341 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2401 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2461 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2521 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2581 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2641 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2701 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2761 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2821 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2881 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2941 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3001 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3061 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3121 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3181 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3241 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3301 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3361 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3421 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3481 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3541 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3601 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3661 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3721 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3781 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3841 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3901 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3961 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4021 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4081 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4141 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
