LOCUS       sequence-118030-        8166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Arms and 3X Flag with P2A Neo for 3' tagging of human 
            RAD21.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  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 8166)
  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_RAD21
FEATURES             Location/Qualifiers
     source          1..8166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      54..642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     543..562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     796..813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1113..1134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1149..1154,1155..1172,1173..1179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1187..1203
                     /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)."
     primer_bind     1192..1214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1211..1227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1211..1227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1404..1606
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1557..1577
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1603..1627
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1703..2833
                     /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"
     CDS             4171..4194
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4198..4221
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4225..4248
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4264..4320
                     /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             4333..5127
                     /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(4387..4406)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4997..5016
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6790..6806)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6799..6821)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(7025..7840)
                     /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
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7748..7767
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7832..7851
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgacggagt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
       61 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      121 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      181 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
      241 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      301 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      361 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      421 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      481 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      541 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      601 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      661 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      721 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      781 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagaagctcg cacattcagc
      841 agcgtttttc agcgcgtttt cgatcaacgt ttcaatgttg gtatcaacac caggtttaac
      901 tttgaactta tcggcactga cggttactga ttttgaactt ttgctttgcc acggaacggt
      961 ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cccaatacgc
     1021 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1081 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     1141 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     1201 caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc
     1261 gactctagag gatccccggg taccgagctc gaattagctt gcatgcctgc aggtcaattc
     1321 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1381 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1441 agcggtttga ctcacgggga tttcaagtct ccaccccatt gacgtcaatg ggagtttgtt
     1501 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     1561 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     1621 tcagatcgcc tggagacgcc atccacgctg ttttgacctc cataggagac accgggaccg
     1681 atccagcctg gggatcccga ccatggcttc gtacccctgc catcaacacg cgtctgcgtt
     1741 cgaccaggct gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg
     1801 gcagcaagaa gccacggaag tccgcctgga gcagaaaatg cccacgctac tgcgggttta
     1861 tatagacggt cctcacggga tggggaaaac caccaccacg caactgctgg tggccctggg
     1921 ttcgcgcgac gatatcgtct acgtacccga gccgatgact tactggcagg tgctgggggc
     1981 ttccgagaca atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc
     2041 ggccggggac gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc
     2101 cgtgaccgac gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc
     2161 gcccccggcc ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc
     2221 ggccgcgcga taccttatgg gcagcatgac cccccaggcc gtgctggcgt tcgtggccct
     2281 catcccgccg accttgcccg gcacaaacat cgtgttgggg gcccttccgg aggacagaca
     2341 catcgaccgc ctggccaaac gccagcgccc cggcgagcgg cttgacctgg ctatgctggc
     2401 cgcgattcgc cgcgtttacg ggctgcttgc caatacggtg cggtatctgc agggcggcgg
     2461 gtcgtggcgg gaggattggg gacagctttc ggggacggcc gtgccgcccc agggtgccga
     2521 gccccagagc aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg
     2581 ggcccccgag ttgctggccc ccaacggcga cctgtacaac gtgtttgcct gggccttgga
     2641 cgtcttggcc aaacgcctcc gtcccatgca cgtctttatc ctggattacg accaatcgcc
     2701 cgccggctgc cgggacgccc tgctgcaact tacctccggg atggtccaga cccacgtcac
     2761 cacccccggc tccataccga cgatctgcga cctggcgcgc acgtttgccc gggagatggg
     2821 ggaggctaac tgaggatccc acgtcactat tgtatactct atattatact ctatgttata
     2881 ctctgtaatc ctactcaata aacgtgtcac gcctgtgaaa ccgtactaag tctcccgtgt
     2941 attcttatca ccatcaggtg acatcctcgc ccaggctgtc aatcatgccg gtatcgattc
     3001 cagtagcacc ggccccacgc tgacaaccca ctcttgcagc gttagcagcg cccctcttaa
     3061 caagccgacc cccaccagcg tcgcggttac taacactcct ctccccgacc tgcagcccag
     3121 cttccccgac ctgcagccca gctgcaaaat gccaagcaag ttgctcaagg tttcttaaaa
     3181 taggacagtt gacatgtgtt tattaatttt ttactttaaa aactataagt gctagatttt
     3241 actactatat tacattagct gtctaaagaa attggttatt ttaataatac atgtcagaac
     3301 aattttgctg gtttcttgga ctttcttatt tggattttag atgtaccata acatactttg
     3361 gccttttacc ctcttgatta acatgagaat cacatactat ttaaaaaagt gcaatctttg
     3421 tcctaagcag aatatatagt ttgttttttc cagtaaccaa aattagcatc tgtgttagtc
     3481 tgttttgtgt tgctctaaag gaatacctga gtctgggtaa tttataaaga agtttatttg
     3541 gctcacagtt ctgcagacta tacaagaagc aaagcaccag catctgcttc tggtgaggcc
     3601 tcagaaagct tatagtcatg gtggaaggca aagggggagc aggtgtgtca catggcaaga
     3661 gagggcatga gagagatggc taggcgcttt taaacaagca gctcttgtgt taactaacga
     3721 gcaggaattc actgattgcc acagggagtg attgataagg gatccaccac cacccccatc
     3781 cccaccccat gacacaaaca cctcactagg ctccatctct aacactggga aattacgttt
     3841 cagcatgaga tttggagggg acaaatattc aaactgtatc agcatcctca ctgaattact
     3901 actttattat acagattaag ttaccctttt ttggttttat ttcagcgtgc tcttgctaaa
     3961 actggagctg aatctatcag tttgcttgag ttatgtcgaa atacgaacag aaaacaagct
     4021 gccgcaaagt tctacagctt cttggttctt aaaaagcagc aagctattga gctgacacag
     4081 gaagaaccgt acagtgacat catcgcaaca cctggaccaa ggttccatat tatagggagc
     4141 ggaggaggtt ccggtggagg tggttctgga gattacaagg atgacgacga taagggcgat
     4201 tacaaggatg acgacgataa gggagattac aaggatgacg acgataaggt ttcaggaagc
     4261 ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct
     4321 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     4381 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     4441 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     4501 ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     4561 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     4621 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     4681 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     4741 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     4801 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     4861 cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     4921 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggccgac
     4981 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     5041 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     5101 tatcgccttc ttgacgagtt cttctgattc gaacatcagc tagaagcatt atagctagtg
     5161 tttgattcac tagtgcttac aaattgcccc catgtgtagg ggacacagaa ccctttgaga
     5221 aaacttagat ttttgtctgt acaaagtctt tgcctttttc cttcttcatt tttttccagt
     5281 acattaaatt tgtcaatttc atctttgagg gaaactgatt agatgggttg tgtttgtgtt
     5341 ctgatggaga aaacagcacc ccaaggactc agaagatgat tttaacagtt cagaacagat
     5401 gtgtgcaata ttggtgcatg taataatgtt gagtggcagt caaaagtcat gatttttatc
     5461 ttagttcttc attactgcat tgaaaaggaa aacctgtctg agaaaatgcc tgacagttta
     5521 atttaaaact atggtgtaag tctttgacaa gaaaaaaaaa caaacaaaca cttctttcca
     5581 tcagtaacac tggcaatctt cctgttaacc actctcctta gggatggtat ctgaaacaac
     5641 aatggtcacc ctcttgagat tcgttttaag tgtaattcca taatgagcag aggtgtacgc
     5701 gaaattgtgt tatgactgat agccttcagc tacaaaaaga taggactgac ctggtttaaa
     5761 gtgttctatt ttgtaaatca ttccatttga gtctttctga tgaacttggc tatactgaaa
     5821 tctgttattt tagtgaggct ccaaaatgag caaagctagg cctgattaga gtagagtgac
     5881 tattaaaaaa cataactttc taggagctat aaatcaaagt tttaaaaaga tgtttggata
     5941 tatttgagta ttccgatcat gaaaacagaa attgccctgc ctactacaag gacagactga
     6001 tgggaaatta tgcacctggt caacttagct tttaagcaga cgatgctgta aaaactaacg
     6061 gcttctctga tatttattgt aagttttagt actgatctcc ttttccagtg ctgcacactc
     6121 ctggtttgga actttaatag cgttgcaacg aaatcctata tccagtttcc tgtaatttaa
     6181 ttgaagaaaa atacatccaa ataaagactt tattattaac agaccagata gcatcagaaa
     6241 tcatgtgact gttatgatta tcagaatgtc ttaacttttt agggcaaagt taacactgaa
     6301 agttctagct taagtgttga aacttttgtg ggaaaaaaaa atcacttttg aaactcagac
     6361 ttcagtgtat acccaataat ttaaaattat gtgaaatgtt ttaaatttgt gaactcgtaa
     6421 ttactgtttt aatgattcag tttcttcaga gtggtaattg tataaaattg ctattgcagc
     6481 tttacattca atatgatgtg cctgtaaacc aaggagtttt ccccgtttgt aaaaagacat
     6541 tgtagataat tgaatgtttg attttagaaa ggtcattagt ttcttgttac acattttgtt
     6601 agtctggttt ttgttgctta tcgggtttaa tattgttctt gaaaatagtt gatgctatgt
     6661 tatgtataac ttttctaata aaagttgtgt tataagctgt acttttttga gtattcatta
     6721 ttcagttggt ggtggtcctt ggaaagtcct ctccactgac tgtagcctcc aattcactgg
     6781 ccgtcgttca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     6841 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     6901 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgcgatt tattcaacaa
     6961 agccgccgtc ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt
     7021 ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     7081 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     7141 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     7201 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     7261 cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag
     7321 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     7381 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     7441 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     7501 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     7561 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     7621 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     7681 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     7741 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     7801 gcggcttcga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     7861 ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac
     7921 atcagagatt ttgagacaca acgtggcttt gttgaataaa tcgaactttt gctgagttga
     7981 aggatcagat cacgcatctt cccgacaacg cagaccgttc cgtggcaaag caaaagttca
     8041 aaatcaccaa ctggtccacc tacaacaaag ctctcatcaa ccgtggctcc ctcactttct
     8101 ggctggatga tggggcgatt caggcctggt atgagtcagc aacaccttct tcacgaggca
     8161 gacctc
//
