LOCUS       sequence-170910-        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for C-terminal tagging with eDHFR trimethoprim regulated degron with
            puro selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  AUTHORS   Sheridan RM, Bentley DL
  TITLE     Selectable one-step PCR-mediated integration of a degron for rapid 
            depletion of endogenous human proteins.
  JOURNAL   Biotechniques. 2016 Feb 1;60(2):69-74. doi: 10.2144/000114378. 
            eCollection 2016 Feb.
  PUBMED    26842351
REFERENCE   2  (bases 1 to 6849)
  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"; doi: "10.2144/000114378";
            journalName: "Biotechniques"; date: "2016-02-1- 1"; volume: "60"; 
            issue: "2"; pages: "69-74"
COMMENT     Sequence Label: pAc5 HA3-eDHFR-T2A-puro
FEATURES             Location/Qualifiers
     source          1..6849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..90
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             97..573
                     /codon_start=1
                     /gene="folA"
                     /product="E. coli dihydrofolate reductase"
                     /label=ecDHFR
                     /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH
                     TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL
                     PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR"
     CDS             580..633
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             658..1257
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(658..677)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1154..1174
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1265..1399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1289..1308)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1343..1362
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(2000..2588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2080..2099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2759..3550,3551..3619))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3382..3401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3620..3724)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3792..3810
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        complement(3962..4037)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4056..4455)
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
ORIGIN
        1 tacccatacg atgttcctga ctatgcgggc tatccctatg acgtcccgga ctatgcagga
       61 tcctatccat atgacgttcc agattacgct gatatcatga tcagtctgat tgcggcgtta
      121 gcggtagatt acgttatcgg catggaaaac gccatgccgt ggaacctgcc tgccgatctc
      181 gcctggttta aacgcaacac cttaaataaa cccgtgatta tgggccgcca tacctgggaa
      241 tcaatcggtc gtccgttgcc aggacgcaaa aatattatcc tcagcagtca accgagtacg
      301 gacgatcgcg taacgtgggt gaagtcggtg gatgaagcca tcgcggcgtg tggtgacgta
      361 ccagaaatca tggtgattgg cggcggtcgc gttattgaac agttcttgcc aaaagcgcaa
      421 aaactgtatc tgacgcatat cgacgcagaa gtggaaggcg acacccattt cccggattac
      481 gagccggatg actgggaatc ggtattcagc gaattccacg atgctgatgc gcagaactct
      541 cacagctatt gctttgagat tctggagcgg cgaaagcttg agggcagagg aagtcttcta
      601 acatgcggtg acgtggagga gaatcccggc cctgctagcg gtagcggcag cggtagcatg
      661 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc
      721 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc
      781 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
      841 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
      901 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
      961 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     1021 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     1081 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     1141 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     1201 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgactc
     1261 gagggatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     1321 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     1381 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     1441 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     1501 tgatcagtcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     1561 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     1621 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     1681 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     1741 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg
     1801 acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta
     1861 tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc
     1921 aggtagatga cgaccatcag ggacagcttc aaggccagca aaaggccagg aaccgtaaaa
     1981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2161 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2401 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     2461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3061 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3121 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3361 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3721 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     3781 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaatta attctatatt
     3841 ctaaaaacac aaatgatact tctaaaaaaa aatcatgaat ggcatcaact ctgaatcaaa
     3901 tctttgcaga tgcacctact tctcatttcc actgtcacat catttttcca gatctaaaaa
     3961 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct
     4021 atttctagct ctaaaacgga agagctcgct cttccgaagt attgaggaaa acatacctat
     4081 ataaatgatc aacatcagga aagagcagtt gagaattata agaattggca aatggtctta
     4141 agaaccctct gcttaagatt ttcaaaattt cctttagaat caaagtgtcc tattgttcgt
     4201 ttgttgaaaa cacagttcga tttattgact ataataaatt gatagtttta aatatagagg
     4261 cacgactaag agagtttggt tttgttttgg ttagggacca aaaaaagtat acataacgaa
     4321 taaaaaagga tttaagtgca aatggtaaaa aagtgtcgag tttttcttga gttgattgtg
     4381 ctgtaatgag actctgcatt cggcatttga ctcggctttt cctactcgtg ccgtatttca
     4441 ggctgcaagt cgaacagatc tcgctgcctg ttatgtggcc cacaaaccaa gacacgtttt
     4501 atggccatta aagctggctg atcgtcgcca aacaccaaat acataatgaa tatgtacaca
     4561 ttcgagaaag aagcgatcaa agaagcgtct tcgggcggag taggagaatg cggaggagaa
     4621 ggagaacgag ctgatctagt atctctccac aatccaatgc caactgacca actggccata
     4681 ttcggagcaa tttgaagcca atttccatcg cctggcgatc gctccattct tggctatatg
     4741 tttttcaccg ttacccgggg ccattttcaa agactcgtcg gcaagataag attgtgtcac
     4801 tcgctgtctc tcttcatttg tcgaagaatg ctgaggaatt tcgcgatgac gtcggcgagt
     4861 attttgaaga atgagaataa tttgtattta tacgaaaatc agttagtgga attttctaca
     4921 aaaacatgtt atctatagat aattttgttg caaaatatgt tgactatgac aaagattgta
     4981 tgtatatctt taatgtattc tcattttctt atgtatttat aatggcaatg atgatactga
     5041 tgatatttta agatgatgcc agaccaaaag gcttgaattt ctgcgtcttt tgccgaacgc
     5101 agtgcatgtg caattgttgt tttttggaat attcaatttt cggactgtcc gctttgattt
     5161 cagtttcttg gcttattcaa aaagcaaagt aaagccaagt ggcggggtct gtggctgggc
     5221 gcgacgtcac cgacgttgga gccactcctt tgaccatgtg tgcgtgtgtg tattattcgt
     5281 gtctcgccac tcgccggttg tttttttctt tttatgctgc gctctctcta gcgccatctc
     5341 gcttacgcat gctcaacgca ccgcatgttg ccgtttcctt ttatgcgtca ttttggctcg
     5401 aaataggcaa ttatttaaac aaagattagt caacgaaaac gctaaaataa ataagtctac
     5461 aatatggtta cttattgcca tgtgtgtgca gccaacgata gcaacaaaag caacaacaca
     5521 ggtggctttc cctctttcac tttttgtttg caagccgcgt gcgagcaaga cggcacgacc
     5581 ggcaaacgca attacgctga caaagagcag acgaagtttt ggcgaaaaac atcaaggcgc
     5641 ctgatacgaa tgcatttgca ataacaattg cgatatttaa tattgtttat gaagctgttt
     5701 gacttcaaaa cacacaaaaa aaaaaataaa acaaattatt tgaaagagaa ttaggaatcg
     5761 gacgcttatc gttagggtaa caacaagaaa tgcttactga gtcacagcct ctggaaaact
     5821 gccgcaagcc agagagagag agaaaaagag ggagagcagc ttagaccgca tgtgcttgtg
     5881 tgtgaggcgt ctctctcttc gtctctgttg cgcaaacgca tagactgcac tgaaaaaatc
     5941 gattacctat tttttatgaa tgaatatttg cactattact attcaaaact attaagatag
     6001 caatcacatt caatagccaa atactatacc acctgagcga tgcaacgaaa tgatcaattt
     6061 gagcaaaaat gctgcatatt taggacggca tcattataga aatgcttctt gctgtgtact
     6121 tttctctcgt ctggcagctg tttcgccgtt attgttaaaa ccggcttaag ttaggtgtgt
     6181 tttctacgac tagtgaatgc cctactagaa gatgtgtgtt gcacaaaatg tccctggaat
     6241 aaccaatttg aagtgcagat agcagtaaac gtaagctaat atgaatatta tttaactgta
     6301 atgttttaat atcgctggac attactaata aacccactat aaacacatgt acatatgtat
     6361 gttttggcat acaatgagta gttggggaaa aaatgtgtaa aagcaccgtg accatcacag
     6421 cataaagata accagctgaa gtatcgaata tgagtaaccc ccaaattgaa tcacatgccg
     6481 caactgatag gacccatgga agtacactct tcatggcgat atacaagaca cacacaagca
     6541 cgaacaccca gttgcggagg aaattctccg taaatgaaaa cccaatcggc gaacaattca
     6601 tacccatata tggtaaaagt tttgaacgcg acttgagagc ggagagcatt gcggctgata
     6661 aggttttagc gctaagcggg ctttataaaa cgggctgcgg gaccagtttt catatcacta
     6721 ccgtttgagt tcttgtgctg tgtggatact cctcccgaca caaagccgct ccatcagcca
     6781 gcagtcgtct aatccagaga ccccggatcg gggtacctac tagtccagtg tggtggaatt
     6841 ggaggcggt
//
