LOCUS       sequence-462118-        4404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of genes by CRISPR/Cas9-mediated homologous 
            recombination; for N-terminal tagging: PAC-3xTy-T2A-3xTy-mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4404)
  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 4404)
  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-PmNG
FEATURES             Location/Qualifiers
     source          1..4404
                     /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             1208..1261
                     /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(2183..2199)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2183..2199)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2196..2218)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2207..2223
                     /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(2231..2237,2238..2255,2256..2261))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2276..2297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2414..2431)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2585..3173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2665..2684)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3344..4135,4136..4204))
                     /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     3967..3986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4205..4309)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4377..4395
                     /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 gaattcatga ctgaatacaa
      481 gccaacggtt cgtcttgcta cgcgagacga tgtgccgcgg gccgtacgga cgctggctgc
      541 tgcgttcgct gattatcccg ccacccgaca tacggttgac ccagatcgac acattgagcg
      601 agtcacagaa cttcaagagt tattcttaac acgggtgggc ttggacattg gtaaggtttg
      661 ggttgcggac gatggcgcgg ctgttgcggt gtggacgacg ccagagtcag tcgaagcggg
      721 cgcagtgttt gctgagatcg gtccccgaat ggcggagctt agcgggtcac gcctcgctgc
      781 acaacagcag atggaaggtc tgttggcacc acatagacct aaggagccgg cttggttctt
      841 agcgacagtt ggcgtcagtc cggaccacca aggtaagggg ctgggttcag cggttgttct
      901 tccaggagta gaggctgcgg aaagggcagg tgtgccggcg ttccttgaaa cgtctgcacc
      961 acgtaacctt cctttttacg agagattggg gtttacagta acagcagatg ttgaagtgcc
     1021 tgaagggccg aggacatggt gcatgacacg taagccggga gccccatggg aggtacacac
     1081 aaaccaggac ccactggacg gctccgaggt gcataccaat caagatccgc tggacggctc
     1141 tgaagttcac accaatcaag atcccttgga tggttctggt agtggttccg gttccggttc
     1201 tcgcgcggag ggtcgtggta gcctgctcac gtgtggtgac gtggaggaga acccaggtcc
     1261 gggtaccgag gtacatacaa atcaggaccc cctcgatggt agtgaagtcc atacaaatca
     1321 ggacccgctg gatggctcag aggttcatac gaaccaagat cctcttgata agcttgtctc
     1381 gaaaggtgag gaagacaata tggccagctt gccagcaacg cacgagcttc atatatttgg
     1441 gtcgatcaac ggtgtggatt tcgacatggt gggtcaaggc acaggtaatc ctaatgacgg
     1501 gtatgaagaa ctcaatttga agagcactaa gggagatctc cagttttccc cgtggattct
     1561 tgtgccgcat attggttatg gttttcatca gtacttacca taccccgatg gaatgtcgcc
     1621 cttccaagca gctatggtgg acggctcggg gtatcaagta catcgcacca tgcaatttga
     1681 ggacggagca tccctcaccg taaactaccg ctatacgtat gaggggagcc acataaaagg
     1741 tgaggctcaa gttaaaggca caggcttccc agctgacggc ccggtcatga ccaattcatt
     1801 aaccgctgct gattggtgcc gttctaagaa gacctaccct aacgacaaga cgatcattag
     1861 tacttttaag tggagttata ccacaggaaa cggaaagcgg tatcgaagca ctgctcgaac
     1921 aacctacaca tttgcgaagc caatggccgc gaactacttg aagaaccagc caatgtatgt
     1981 gtttcgaaaa acggaactga agcattcgaa aaccgagctt aattttaaag agtggcagaa
     2041 ggcgtttaca gacgtgatgg ggatggacga attgtataag ggatccggat caggatctgg
     2101 atcaggatcg ggtagtgagc tcctgcaggg atgccgggac cgacgagtgc agaggcgtgc
     2161 aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2221 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2281 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2341 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2401 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2461 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2521 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2581 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2641 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2701 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2761 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2821 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2881 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2941 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3001 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3061 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3121 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3181 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3241 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3301 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3361 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3421 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3481 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3541 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3601 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3661 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3721 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3781 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3841 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3901 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3961 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4021 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4081 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4141 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4201 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4261 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4321 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     4381 ggcgtatcac gaggcccttt cgtc
//
