LOCUS       sequence-118028-        7405 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Arms and 3X Flag with P2A Neo for 3' tagging of human 
            GABPA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7405)
  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 7405)
  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_GABPA
FEATURES             Location/Qualifiers
     source          1..7405
                     /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             4179..4202
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4206..4229
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4233..4256
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4272..4328
                     /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             4341..5135
                     /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(4395..4414)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5005..5024
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6029..6045)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6038..6060)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(6264..7079)
                     /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     6987..7006
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7071..7090
                     /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 gctgtctctt tgcaatgtcc tttacctgtt tttctgtatc
     3181 ctctgtttct tttagacctt ttccttagac ttaaagctcc ctattgggct tggataattt
     3241 atctgaaatt tggaaagcgt ttacatagtg aaataataaa gatgaagaca tgacttcatc
     3301 atataggatt ctttacaaga cagaaaagaa gtggaatata tgtattaatt ttttttatat
     3361 tatcgatttt tattaacata gcttatattt tgtagagggc tgtgtgccag tcattgtata
     3421 ctaagcactt tacaaagatt atccctttaa tcctcaacaa tgatatgagg gtataagcac
     3481 tgttaacctc actttataga tgaggaataa aagctttgag aaaatatgac tcctgtggtc
     3541 tcaaaaatgt taggttttga atcttggctt tctgactcca aagctcttgt tcttaggctc
     3601 ttctactgct ctcaaggttt tttgttttgt ttaataatta tttttaaatc aatgttttat
     3661 tctaattgtt gaaaatatgt tttgaaagtt taatgtatag agttttggag tagtgagcat
     3721 tgaaatttta gtaaataatg ttgggatgtt tcatttgata gtagcattaa atattctaca
     3781 tagcttcaat attaattgct attgtggcgg atgcagtact ctactaggct tctgcttatg
     3841 gaatagtctg taaattattg taagaagtct catttatttt ttctgaagtc tctaattttt
     3901 ttttctcttt ttgaagatat tattacgatg gggacatgat ttgtaaagtt caaggcaaga
     3961 gatttgtgta caagtttgtc tgtgacttga agactcttat tggatacagt gcagcggagt
     4021 tgaaccgttt ggtcacagaa tgtgaacaga agaaacttgc aaagatgcag ctccatggaa
     4081 ttgcccagcc agtcacagca gtagctctgg ctactgcttc tctgcaaacg gaaaaggata
     4141 atgggagcgg aggaggttcc ggtggaggtg gttctggaga ttacaaggat gacgacgata
     4201 agggcgatta caaggatgac gacgataagg gagattacaa ggatgacgac gataaggttt
     4261 caggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc
     4321 ctggacctgg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4381 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4441 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4501 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4561 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4621 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4681 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4741 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4801 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4861 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4921 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4981 tggccgaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     5041 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5101 tcgccttcta tcgccttctt gacgagttct tctgattcga acatcactcc aaagtctttc
     5161 ttaaaatgtt tagagcaagt atagctctta cctttattac tgaatttgaa tcttctttta
     5221 tttctaggct gtacagtctg atgcatgatt tttttataaa tatttcatac tcttgtgaat
     5281 ttggatcttt ttactttgag catatatttt agaatatgtg tatgttaaag gatctccaca
     5341 atgtctgcag tgtgaaggca ggttcattgt ggaatagttt aacagtcagg aaggctaaac
     5401 tggtcagtat taatgtgtag ccctaccaaa aatagccagt agtatctgaa aatgaaaaat
     5461 aaatgaagta tctctaggaa acagtctggc ttaactattt ttgaaaatat aactgtttcc
     5521 cctctctgct gctttagatg ttgctttaca tagaaccaga aaatggaatt tctcagctaa
     5581 agcatgtgtg cctgtttcat ctaatcaagc agagctaaaa tgttcatacc gaataaattt
     5641 atattaataa attactaaac taagagtatc aggttattta tatatttgca agcaaaggac
     5701 agtaagaagt tgactggcaa aagagcagtg ctgaaggagg agatccaggt ttaaatctgg
     5761 cttattaact caagccaatt ttaaggattt tctgtataga ttactcatgt cagaccaaga
     5821 atttaaatta ttttgagaga ggcatttaat tctaataaac cagctgttat aaaaattata
     5881 aaatgatctc tgtttttcct gtcagagatt taaaaaactg aaaaggtata cctcaaccca
     5941 aaaataaagg tttgttttgg tttgttatgg cttcctcttg gaaagtcctc tccactgact
     6001 gtagcctcca attcactggc cgtcgttcac tggccgtcgt tttacaacgt cgtgactggg
     6061 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     6121 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     6181 aatgcgattt attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct ctgccagtgt
     6241 tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     6301 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     6361 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     6421 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     6481 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     6541 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     6601 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     6661 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     6721 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     6781 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     6841 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     6901 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     6961 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     7021 tccatgttgg aatttaatcg cggcttcgag caagacgttt cccgttgaat atggctcata
     7081 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     7141 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat
     7201 cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc
     7261 gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac
     7321 cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca
     7381 acaccttctt cacgaggcag acctc
//
