LOCUS       sequence-147903-        6969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for 3' FLAG tag of human ATF4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6969)
  TITLE     Mendenhall CRISPR tagging plasmids
REFERENCE   2  (bases 1 to 6969)
  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"
COMMENT     Sequence Label: pFETCh_ATF4
FEATURES             Location/Qualifiers
     source          1..6969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             459..476
                     /codon_start=1
                     /product="N-myristoylation signal from Src kinase (Pellman 
                     et al., 1985; Kaplan et al., 1988)"
                     /label=myr
                     /translation="GSSKSK"
     CDS             745..768
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             772..795
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             799..822
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             838..894
                     /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             907..1701
                     /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(961..980)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1571..1590
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2472..2488)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2481..2503)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(2706..3521)
                     /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     3429..3448
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3513..3532
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3901..4489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4390..4409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4643..4660
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4960..4981
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4996..5001,5002..5019,5020..5026)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5034..5050
                     /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     5039..5061
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5058..5074
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5058..5074
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5251..5453
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5404..5424
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5450..5474
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5550..6680
                     /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"
ORIGIN
        1 ttggatgaca cttgtgatct ctttgccccc ctagtccagg agactaataa gcagcccccc
       61 cagacggtga acccaattgg ccatctccca gaaagtttaa caaaacccga ccaggttgcc
      121 cccttcacct tcttacaacc tcttcccctt tccccagggg tcctgtcctc cactccagat
      181 cattccttta gtttagagct gggcagtgaa gtggatatca ctgaaggaga taggaagcca
      241 gactacactg cttacgttgc catgatccct cagtgcataa aggaggaaga caccccttca
      301 gataatgata gtggcatctg tatgagccca gagtcctatc tggggtctcc tcagcacagc
      361 ccctctacca ggggctctcc aaataggagc ctcccatctc caggtgttct ctgtgggtct
      421 gcccgtccca aaccttacga tcctcctgga gagaagatgg tagcagcaaa agtaaagggt
      481 gagaaactgg ataagaagct gaaaaaaatg gagcaaaaca agacagcagc cactaggtac
      541 cgccagaaga agagggcgga gcaggaggct cttactggtg agtgcaaaga gctggaaaag
      601 aagaacgagg ctctaaaaga gagggcggat tccctggcca aggagatcca gtacctgaaa
      661 gatttgatag aagaggtccg caaggcaagg gggaagaaaa gggtccccgg gagcggagga
      721 ggttccggtg gaggtggttc tggagattac aaggatgacg acgataaggg cgattacaag
      781 gatgacgacg ataagggaga ttacaaggat gacgacgata aggtttcagg aagcggagct
      841 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctggatcg
      901 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
      961 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     1021 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     1081 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     1141 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     1201 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     1261 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     1321 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     1381 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     1441 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     1501 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc cgaccgctat
     1561 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     1621 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     1681 cttcttgacg agttcttctg attcgaacat cgtcaggagc gtcaatgtgc ttgtacatag
     1741 agtgctgtag ctgtgtgttc caataaatta ttttgtaggg aaagtacttg tgcgtttgaa
     1801 ttcctttcct ggcaaagtaa ccgagtgaag cgccacccac ccaccccaag ctggtcttaa
     1861 catttcctgt gagatctggg ggaggaggca gtccaaggct gaagatcggt tcccagagtg
     1921 ggatttgctc atccctgctc ggtgtaggcc acgatggctg attcatgggg ccctggagcc
     1981 gtatagttgg ccatttatag taagctgggt cctttgccca gagctgagtg ctggttgtgt
     2041 agggggagga ggctggagat ctctgggaga ttcagaggga gggacagcct gggcctccac
     2101 ctgtttattc aagagggctt catgccaaca gctgtcgcat aaaaccctga ataaggttgc
     2161 acggtctttg agatagcaca caagattagg tttcacttgt tacctgaaca gttctgcaga
     2221 agcctcatcc tgaagaaaga aatggctcct tttgggaatt gatgctgaaa caggacagga
     2281 gctgagggtt ggcctccgtt tctccttacg gggtggaagg atgacagcct gcgcaggttc
     2341 tggctagagc tgggcacaaa ggcaggcaac agttcctgtg aaggctgttg tgcctgctgc
     2401 tatctaaact aggaggcctc ttggaaagtc ctctccactg actgtagcct ccaattcact
     2461 ggccgtcgtt cactggccgt cgttttacaa cgtcgtgact gggaaaccct ggcgttaccc
     2521 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2581 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgcgat ttattcaaca
     2641 aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat
     2701 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     2761 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     2821 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2881 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2941 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     3001 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     3061 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     3121 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     3181 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     3241 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     3301 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     3361 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     3421 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     3481 cgcggcttcg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     3541 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     3601 catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg
     3661 aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc
     3721 aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc cctcactttc
     3781 tggctggatg atggggcgat tcaggcctgg tatgagtcag caacaccttc ttcacgaggc
     3841 agacctctcg acggagttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3901 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3961 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     4021 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     4081 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     4141 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     4201 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     4261 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     4321 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     4381 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     4441 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     4501 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     4561 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     4621 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag aagctcgcac
     4681 attcagcagc gtttttcagc gcgttttcga tcaacgtttc aatgttggta tcaacaccag
     4741 gtttaacttt gaacttatcg gcactgacgg ttactgattt tgaacttttg ctttgccacg
     4801 gaacggtctg cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttccc
     4861 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     4921 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     4981 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5041 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc ttgcatgcct
     5101 gcaggtcgac tctagaggat ccccgggtac cgagctcgaa ttagcttgca tgcctgcagg
     5161 tcaattccct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     5221 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     5281 cgtggatagc ggtttgactc acggggattt caagtctcca ccccattgac gtcaatggga
     5341 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     5401 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
     5461 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat aggagacacc
     5521 gggaccgatc cagcctgggg atcccgacca tggcttcgta cccctgccat caacacgcgt
     5581 ctgcgttcga ccaggctgcg cgttctcgcg gccatagcaa ccgacgtacg gcgttgcgcc
     5641 ctcgccggca gcaagaagcc acggaagtcc gcctggagca gaaaatgccc acgctactgc
     5701 gggtttatat agacggtcct cacgggatgg ggaaaaccac caccacgcaa ctgctggtgg
     5761 ccctgggttc gcgcgacgat atcgtctacg tacccgagcc gatgacttac tggcaggtgc
     5821 tgggggcttc cgagacaatc gcgaacatct acaccacaca acaccgcctc gaccagggtg
     5881 agatatcggc cggggacgcg gcggtggtaa tgacaagcgc ccagataaca atgggcatgc
     5941 cttatgccgt gaccgacgcc gttctggctc ctcatatcgg gggggaggct gggagctcac
     6001 atgccccgcc cccggccctc accctcatct tcgaccgcca tcccatcgcc gccctcctgt
     6061 gctacccggc cgcgcgatac cttatgggca gcatgacccc ccaggccgtg ctggcgttcg
     6121 tggccctcat cccgccgacc ttgcccggca caaacatcgt gttgggggcc cttccggagg
     6181 acagacacat cgaccgcctg gccaaacgcc agcgccccgg cgagcggctt gacctggcta
     6241 tgctggccgc gattcgccgc gtttacgggc tgcttgccaa tacggtgcgg tatctgcagg
     6301 gcggcgggtc gtggcgggag gattggggac agctttcggg gacggccgtg ccgccccagg
     6361 gtgccgagcc ccagagcaac gcgggcccac gaccccatat cggggacacg ttatttaccc
     6421 tgtttcgggc ccccgagttg ctggccccca acggcgacct gtacaacgtg tttgcctggg
     6481 ccttggacgt cttggccaaa cgcctccgtc ccatgcacgt ctttatcctg gattacgacc
     6541 aatcgcccgc cggctgccgg gacgccctgc tgcaacttac ctccgggatg gtccagaccc
     6601 acgtcaccac ccccggctcc ataccgacga tctgcgacct ggcgcgcacg tttgcccggg
     6661 agatggggga ggctaactga ggatcccacg tcactattgt atactctata ttatactcta
     6721 tgttatactc tgtaatccta ctcaataaac gtgtcacgcc tgtgaaaccg tactaagtct
     6781 cccgtgtatt cttatcacca tcaggtgaca tcctcgccca ggctgtcaat catgccggta
     6841 tcgattccag tagcaccggc cccacgctga caacccactc ttgcagcgtt agcagcgccc
     6901 ctcttaacaa gccgaccccc accagcgtcg cggttactaa cactcctctc cccgacctgc
     6961 agcccagct
//
