LOCUS       sequence-118029-        7221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology Arms and 3X Flag with P2A Neo for 3' tagging of human ATF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7221)
  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 7221)
  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_ATF1
FEATURES             Location/Qualifiers
     source          1..7221
                     /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             4022..4045
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4049..4072
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4076..4099
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4115..4171
                     /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             4184..4978
                     /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(4238..4257)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4848..4867
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5845..5861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5854..5876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(6080..6895)
                     /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     6803..6822
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6887..6906
                     /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 gcttccccga cctgcagccc agcttccccg acctgcagcc
     3181 cagctagaac tgagtatttg ctcaagcatt gtttgtaatt tgaacttaaa taaaagaagt
     3241 catgtgtcag aagttaacca gaggacccat aatttcactc tttggtattt attcaagaga
     3301 aatgaaacct ttctacaaaa agactggcac aaaaacatgc ctatgcagct aaccctagaa
     3361 acatcgcaag agactggata cacaaatggt ggttatattc atacagtgaa ctatgtccag
     3421 cagtgtgaag gaacaaaccc ccatgcagca acatggatga acctcagatg ggctgagtga
     3481 agggacacca gagtacatag tgtatgacac tcttacatga ggttctagaa taggaaaaaa
     3541 actaatctct ggtgataaaa atcaaaacga tagttgcttg gaagggggct gaccaggaag
     3601 gggcatgagg gaactttctg ggacagtggg aatgttctgt tgtcttgaca gaggtgtggg
     3661 ttatacaggt atacacaatt gccaaaactg actgaattct acacttaaga tctatgtatt
     3721 ctctgtgtgt agatgatact ttaaagagaa aaaaaggtga ccacggaaaa ttactgaaga
     3781 acatttacca tttaaagaat gtgaagtttt ttctaacatt gtttttttaa tgctcatatt
     3841 tagagaagct gctcgagaat gtcgcagaaa gaagaaagaa tatgtgaaat gcctggaaaa
     3901 ccgagttgca gtcctggaaa atcaaaataa aactctaata gaagagttaa aaactttgaa
     3961 ggatctttat tccaacaagt ccgtggggag cggaggaggt tccggtggag gtggttctgg
     4021 agattacaag gatgacgacg ataagggcga ttacaaggat gacgacgata agggagatta
     4081 caaggatgac gacgataagg tttcaggaag cggagctact aacttcagcc tgctgaagca
     4141 ggctggagac gtggaggaga accctggacc tggatcgttt cgcatgattg aacaagatgg
     4201 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     4261 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     4321 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     4381 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     4441 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     4501 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4561 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4621 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4681 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     4741 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4801 catcgactgt ggccggctgg gtgtggccga ccgctatcag gacatagcgt tggctacccg
     4861 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4921 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgatt
     4981 cgaacatctc ctaagaaaga aaatattttt gtggacatgc ataaaaatta aatggatttc
     5041 ctagtggagt tttataaatt aaaaggtcaa aactgaagct ttttatttag gcttttccaa
     5101 atcaaggata aatatcttac gcacgatatc tagtgacaga ggagaaagtg gaaaatgacc
     5161 tcaaggaagc tacgggcaca actggaagcc ttgtagaaat taaacatatt caaggagcaa
     5221 gaaatgaact ttcagcagtc taaattttct aaataaccaa tagttgccaa tctaaaatgg
     5281 cagagaagat gaaatttgat aaactgaatt ttttttaaaa atccatttac cctacaggtt
     5341 tgcatttgtt tgctgaaatt tacctttttt tagttatata tatgtgtgtg tgtgtgtgta
     5401 atttctgcca ataaattcta aattacaaag gtaagagaaa acctagtaca ttactaaata
     5461 tataaagtat atgttctgat tatgtatact tgttctagtg tcaagtcttt ttaagtgggt
     5521 ttttaaaagt ttgttattgg acttgaatgg atttttgaga ctaggttaat tatttttgag
     5581 gtcttatcct aaaaggcatc taaggtacat gaatggagta tggtgatttt ataacatttt
     5641 ttatcagaat ggaaaaagaa ctgtttaaaa gtttgatact tttaaatagt tggttttttt
     5701 gcttactctg gtaatgattt tctacaaata cataataaat tgtttttttg agtctatatt
     5761 ctgtatgcag ttgaatatcc attacttatt ctcttggaaa gtcctctcca ctgactgtag
     5821 cctccaattc actggccgtc gttcactggc cgtcgtttta caacgtcgtg actgggaaaa
     5881 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     5941 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     6001 cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca
     6061 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     6121 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     6181 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     6241 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     6301 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     6361 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     6421 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     6481 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     6541 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     6601 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     6661 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     6721 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     6781 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     6841 tgttggaatt taatcgcggc ttcgagcaag acgtttcccg ttgaatatgg ctcataacac
     6901 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     6961 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa
     7021 cttttgctga gttgaaggat cagatcacgc atcttcccga caacgcagac cgttccgtgg
     7081 caaagcaaaa gttcaaaatc accaactggt ccacctacaa caaagctctc atcaaccgtg
     7141 gctccctcac tttctggctg gatgatgggg cgattcaggc ctggtatgag tcagcaacac
     7201 cttcttcacg aggcagacct c
//
