LOCUS       sequence-252696-        5595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3X Flag with P2A-Neo for 3' tagging of endogenous proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5595)
  AUTHORS   Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts 
            BS, Mackiewicz M, Mendenhall EM, Myers RM
  TITLE     CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.
  JOURNAL   Genome Res. 2015 Sep 9.
  PUBMED    26355004
REFERENCE   2  (bases 1 to 5595)
  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: "Genome Res.
            2015 Sep 9."
COMMENT     Sequence Label: pFETCh_Donor (EMM0021)
FEATURES             Location/Qualifiers
     source          1..5595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(218..235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(389..977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(469..488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1346..1365)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1357..2172
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNILQFHLMLDEFF"
     primer_bind     complement(1430..1449)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2376..2398
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2391..2407
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(2492..3286)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2603..2622)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3213..3232
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3299..3355)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(3371..3394)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3398..3421)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3425..3448)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3793..4923)
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(4999..5023)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(5020..5222)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5049..5069)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(5399..5415)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5399..5415)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5412..5434)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5423..5439
                     /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(5447..5453,5454..5471,5472..5477))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5492..5513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaggcggttt gcgtattggg aacttttgct gagttgaagg atcagatcac gcatcttccc
       61 gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa tcagtaaccg tcagtgccga
      121 taagttcaaa gttaaacctg gtgttgatac caacattgaa acgttgatcg aaaacgcgct
      181 gaaaaacgct gctgaatgtg cgagcttctt ccgcttcctc gctcactgac tcgctgcgct
      241 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
      301 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
      361 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      421 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
      481 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
      541 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
      601 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      661 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      721 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
      781 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
      841 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
      901 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
      961 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     1021 actccgtcga gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc
     1081 gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt gttgtaggtg
     1141 gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga
     1201 tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag ccacgttgtg
     1261 tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa
     1321 ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg
     1381 tcttgctcga agccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1441 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat
     1501 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1561 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     1621 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag
     1681 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     1741 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     1801 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     1861 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc
     1921 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     1981 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2041 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2101 caaaaatatg gtattgataa tcctgatatg aatatattgc agtttcattt gatgctcgat
     2161 gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact
     2221 tgacgggacg gcggctttgt tgaataaatc gcattcgcca ttcaggctgc gcaactgttg
     2281 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     2341 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     2401 ggccagtgaa cgacggccag tgaattggag gctacagtca gtggagagga ctttccaaga
     2461 tgtcttcgta taagaagaca tgatgttcga atcagaagaa ctcgtcaaga aggcgataga
     2521 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     2581 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     2641 cggccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     2701 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg
     2761 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     2821 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     2881 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     2941 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     3001 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     3061 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     3121 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     3181 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     3241 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatccag
     3301 gtccagggtt ctcctccacg tctccagcct gcttcagcag gctgaagtta gtagctccgc
     3361 ttcctgaaac cttatcgtcg tcatccttgt aatctccctt atcgtcgtca tccttgtaat
     3421 cgcccttatc gtcgtcatcc ttgtaatctc cagaaccacc tccaccggaa cctcctccgc
     3481 tcagagaccc gcgtaaggtc tcaagctggg ctgcaggtcg gggagaggag tgttagtaac
     3541 cgcgacgctg gtgggggtcg gcttgttaag aggggcgctg ctaacgctgc aagagtgggt
     3601 tgtcagcgtg gggccggtgc tactggaatc gataccggca tgattgacag cctgggcgag
     3661 gatgtcacct gatggtgata agaatacacg ggagacttag tacggtttca caggcgtgac
     3721 acgtttattg agtaggatta cagagtataa catagagtat aatatagagt atacaatagt
     3781 gacgtgggat cctcagttag cctcccccat ctcccgggca aacgtgcgcg ccaggtcgca
     3841 gatcgtcggt atggagccgg gggtggtgac gtgggtctgg accatcccgg aggtaagttg
     3901 cagcagggcg tcccggcagc cggcgggcga ttggtcgtaa tccaggataa agacgtgcat
     3961 gggacggagg cgtttggcca agacgtccaa ggcccaggca aacacgttgt acaggtcgcc
     4021 gttgggggcc agcaactcgg gggcccgaaa cagggtaaat aacgtgtccc cgatatgggg
     4081 tcgtgggccc gcgttgctct ggggctcggc accctggggc ggcacggccg tccccgaaag
     4141 ctgtccccaa tcctcccgcc acgacccgcc gccctgcaga taccgcaccg tattggcaag
     4201 cagcccgtaa acgcggcgaa tcgcggccag catagccagg tcaagccgct cgccggggcg
     4261 ctggcgtttg gccaggcggt cgatgtgtct gtcctccgga agggccccca acacgatgtt
     4321 tgtgccgggc aaggtcggcg ggatgagggc cacgaacgcc agcacggcct ggggggtcat
     4381 gctgcccata aggtatcgcg cggccgggta gcacaggagg gcggcgatgg gatggcggtc
     4441 gaagatgagg gtgagggccg ggggcggggc atgtgagctc ccagcctccc ccccgatatg
     4501 aggagccaga acggcgtcgg tcacggcata aggcatgccc attgttatct gggcgcttgt
     4561 cattaccacc gccgcgtccc cggccgatat ctcaccctgg tcgaggcggt gttgtgtggt
     4621 gtagatgttc gcgattgtct cggaagcccc cagcacctgc cagtaagtca tcggctcggg
     4681 tacgtagacg atatcgtcgc gcgaacccag ggccaccagc agttgcgtgg tggtggtttt
     4741 ccccatcccg tgaggaccgt ctatataaac ccgcagtagc gtgggcattt tctgctccag
     4801 gcggacttcc gtggcttctt gctgccggcg agggcgcaac gccgtacgtc ggttgctatg
     4861 gccgcgagaa cgcgcagcct ggtcgaacgc agacgcgtgt tgatggcagg ggtacgaagc
     4921 catggtcggg atccccaggc tggatcggtc ccggtgtctc ctatggaggt caaaacagcg
     4981 tggatggcgt ctccaggcga tctgacggtt cactaaacga gctctgctta tatagacctc
     5041 ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg
     5101 aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact
     5161 tgaaatcccc gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc
     5221 accatggtaa tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag
     5281 gtcatgtact gggcataatg ccagggaatt gacctgcagg catgcaagct aattcgagct
     5341 cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt
     5401 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5461 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5521 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5581 gccaacgcgc gggga
//
