LOCUS       sequence-463540-        4184 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-mScarlet.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4184)
  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 4184)
  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-BmSc
FEATURES             Location/Qualifiers
     source          1..4184
                     /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(1963..1979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1963..1979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1976..1998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1987..2003
                     /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(2011..2017,2018..2035,2036..2041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2056..2077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2194..2211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2365..2953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2445..2464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3124..3915,3916..3984))
                     /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     3747..3766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3985..4089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4157..4175
                     /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 atcaaagggc gaagcagtaa tcaaggagtt
     1201 catgcggttt aaggtacaca tggagggttc catgaatggc catgaatttg agatcgaagg
     1261 agagggggaa gggcgtcctt acgaaggaac acaaacagct aaacttaagg taactaaggg
     1321 gggacccctc cccttttcct gggacatact gtcaccacaa ttcatgtacg gcagccgagc
     1381 ctttataaaa catccagctg atatacccga ttattataag caaagttttc ccgagggctt
     1441 taagtgggaa agggtcatga acttcgagga cgggggtgcg gtaactgtaa cccaagacac
     1501 ctctctcgaa gacgggacgc ttatttataa ggtgaaactc cgggggacca attttccccc
     1561 tgacggacct gtcatgcaga agaagacaat gggctgggag gcgtccacag agcgcctgta
     1621 tccggaggat ggcgtactta agggtgacat aaaaatggca cttcgtttga aggacggtgg
     1681 ccgttatctc gctgacttta agacaactta taaggcgaaa aagccggtac agatgccagg
     1741 agcatacaat gttgaccgca aattggacat caccagccac aacgaagact ataccgtcgt
     1801 tgaacagtac gaacggagtg aagggaggca cagtacaggc gggatggacg aattgtataa
     1861 aggatccgga tcaggatctg gatcaggatc gggtagtgag ctcctgcagg atgccgggac
     1921 cgacgagtgc agaggcgtgc aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     1981 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2041 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2101 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2161 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2221 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2281 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2341 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2401 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2461 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2521 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2581 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2641 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2701 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2761 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2821 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2881 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2941 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3001 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3061 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3121 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3181 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3241 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3301 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3361 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3421 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3481 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3541 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3601 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3661 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3721 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3781 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3841 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3901 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3961 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4021 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4081 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     4141 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
