LOCUS       sequence-170909-        7112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for C-terminal tagging with eDHFR trimethoprim regulated degron with
            neo selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7112)
  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 7112)
  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-neo
FEATURES             Location/Qualifiers
     source          1..7112
                     /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             640..1434
                     /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(694..713)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1304..1323
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1442..1576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1466..1485)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1520..1539
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(2177..2765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2257..2276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2936..3727,3728..3796))
                     /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     3559..3578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3797..3901)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3969..3987
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        complement(4139..4214)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4233..4632)
                     /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 cctgctagca tgattgaaca agatggattg
      661 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
      721 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
      781 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
      841 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
      901 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
      961 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     1021 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     1081 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     1141 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     1201 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     1261 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     1321 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     1381 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgactcgag
     1441 ggatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     1501 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     1561 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     1621 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     1681 tcagtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     1741 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     1801 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     1861 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     1921 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg
     1981 cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga
     2041 ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg
     2101 tagatgacga ccatcaggga cagcttcaag gccagcaaaa ggccaggaac cgtaaaaagg
     2161 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2221 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2281 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2341 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2401 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2461 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2521 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2581 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     2641 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2701 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2761 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     2821 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     2881 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     2941 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3001 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3061 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3121 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3181 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3241 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3301 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     3361 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     3421 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     3481 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     3541 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     3601 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     3661 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     3721 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     3781 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     3841 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     3901 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     3961 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt ctatattcta
     4021 aaaacacaaa tgatacttct aaaaaaaaat catgaatggc atcaactctg aatcaaatct
     4081 ttgcagatgc acctacttct catttccact gtcacatcat ttttccagat ctaaaaaagc
     4141 accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt
     4201 tctagctcta aaacggaaga gctcgctctt ccgaagtatt gaggaaaaca tacctatata
     4261 aatgatcaac atcaggaaag agcagttgag aattataaga attggcaaat ggtcttaaga
     4321 accctctgct taagattttc aaaatttcct ttagaatcaa agtgtcctat tgttcgtttg
     4381 ttgaaaacac agttcgattt attgactata ataaattgat agttttaaat atagaggcac
     4441 gactaagaga gtttggtttt gttttggtta gggaccaaaa aaagtataca taacgaataa
     4501 aaaaggattt aagtgcaaat ggtaaaaaag tgtcgagttt ttcttgagtt gattgtgctg
     4561 taatgagact ctgcattcgg catttgactc ggcttttcct actcgtgccg tatttcaggc
     4621 tgcaagtcga acagatctcg ctgcctgtta tgtggcccac aaaccaagac acgttttatg
     4681 gccattaaag ctggctgatc gtcgccaaac accaaataca taatgaatat gtacacattc
     4741 gagaaagaag cgatcaaaga agcgtcttcg ggcggagtag gagaatgcgg aggagaagga
     4801 gaacgagctg atctagtatc tctccacaat ccaatgccaa ctgaccaact ggccatattc
     4861 ggagcaattt gaagccaatt tccatcgcct ggcgatcgct ccattcttgg ctatatgttt
     4921 ttcaccgtta cccggggcca ttttcaaaga ctcgtcggca agataagatt gtgtcactcg
     4981 ctgtctctct tcatttgtcg aagaatgctg aggaatttcg cgatgacgtc ggcgagtatt
     5041 ttgaagaatg agaataattt gtatttatac gaaaatcagt tagtggaatt ttctacaaaa
     5101 acatgttatc tatagataat tttgttgcaa aatatgttga ctatgacaaa gattgtatgt
     5161 atatctttaa tgtattctca ttttcttatg tatttataat ggcaatgatg atactgatga
     5221 tattttaaga tgatgccaga ccaaaaggct tgaatttctg cgtcttttgc cgaacgcagt
     5281 gcatgtgcaa ttgttgtttt ttggaatatt caattttcgg actgtccgct ttgatttcag
     5341 tttcttggct tattcaaaaa gcaaagtaaa gccaaaaaag cgagatggca ataccaaatg
     5401 cggcaaaacg gtagtggaag gaaaggggtg cggggcagcg gaaggaaggg tggggcgggg
     5461 cgtggcgggg tctgtggctg ggcgcgacgt caccgacgtt ggagccactc ctttgaccat
     5521 gtgtgcgtgt gtgtattatt cgtgtctcgc cactcgccgg ttgttttttt ctttttatgc
     5581 tgcgctctct ctagcgccat ctcgcttacg catgctcaac gcaccgcatg ttgccgtttc
     5641 cttttatgcg tcattttggc tcgaaatagg caattattta aacaaagatt agtcaacgaa
     5701 aacgctaaaa taaataagtc tacaatatgg ttacttattg ccatgtgtgt gcagccaacg
     5761 atagcaacaa aagcaacaac acaggtggct ttccctcttt cactttttgt ttgcaagccg
     5821 cgtgcgagca agacggcacg accggcaaac gcaattacgc tgacaaagag cagacgaagt
     5881 tttggcgaaa aacatcaagg cgcctgatac gaatgcattt gcaataacaa ttgcgatatt
     5941 taatattgtt tatgaagctg tttgacttca aaacacacaa aaaaaaaaat aaaacaaatt
     6001 atttgaaaga gaattaggaa tcggacgctt atcgttaggg taacaacaag aaatgcttac
     6061 tgagtcacag cctctggaaa actgccgcaa gccagagaga gagagaaaaa gagggagagc
     6121 agcttagacc gcatgtgctt gtgtgtgagg cgtctctctc ttcgtctctg ttgcgcaaac
     6181 gcatagactg cactgaaaaa atcgattacc tattttttat gaatgaatat ttgcactatt
     6241 actattcaaa actattaaga tagcaatcac attcaatagc caaatactat accacctgag
     6301 cgatgcaacg aaatgatcaa tttgagcaaa aatgctgcat atttaggacg gcatcattat
     6361 agaaatgctt cttgctgtgt acttttctct cgtctggcag ctgtttcgcc gttattgtta
     6421 aaaccggctt aagttaggtg tgttttctac gactagtgaa tgccctacta gaagatgtgt
     6481 gttgcacaaa atgtccctgg aataaccaat ttgaagtgca gatagcagta aacgtaagct
     6541 aatatgaata ttatttaact gtaatgtttt aatatcgctg gacattacta ataaacccac
     6601 tataaacaca tgtacatatg tatgttttgg catacaatga gtagttgggg aaaaaatgtg
     6661 taaaagcacc gtgaccatca cagcataaag ataaccagct gaagtatcga atatgagtaa
     6721 cccccaaatt gaatcacatg ccgcaactga taggacccat ggaagtacac tcttcatggc
     6781 gatatacaag acacacacaa gcacgaacac ccagttgcgg aggaaattct ccgtaaatga
     6841 aaacccaatc ggcgaacaat tcatacccat atatggtaaa agttttgaac gcgacttgag
     6901 agcggagagc attgcggctg ataaggtttt agcgctaagc gggctttata aaacgggctg
     6961 cgggaccagt tttcatatca ctaccgtttg agttcttgtg ctgtgtggat actcctcccg
     7021 acacaaagcc gctccatcag ccagcagtcg tctaatccag agaccccgga tcggggtacc
     7081 tactagtcca gtgtggtgga attggaggcg gt
//
