LOCUS       sequence-248874-        6406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides the repair template with PP2Ac microhomology on each end 
            for PP2Ac N-ter H3FAID tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6406)
  AUTHORS   Lin DW, Chung BP, Huang JW, Wang X, Huang L, Kaiser P
  TITLE     Microhomology based CRISPR tagging tools for protein tracking, 
            purification, and depletion.
  JOURNAL   J Biol Chem. 2019 May 28. pii: RA119.008422. doi: 
            10.1074/jbc.RA119.008422.
  PUBMED    31138654
REFERENCE   2  (bases 1 to 6406)
  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: "J Biol 
            Chem. 2019 May 28. pii: RA119.008422. doi: 
            10.1074/jbc.RA119.008422."
COMMENT     Sequence Label: pN-PITCh-H3FAID
FEATURES             Location/Qualifiers
     source          1..6406
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /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)."
     regulatory      1587..1596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1593..1595,1596..1598,1599..2309)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1593..1595,1596..1598,1599..2309)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1638..1659)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1899..1918)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2246..2267
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2319..2372
                     /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             2373..2969
                     /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(2373..2392)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2869..2889
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2979..3035
                     /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             3048..3065
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3066..3137
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             3144..3347
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     primer_bind     complement(3421..3438)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3427..3538
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3656..3676)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3661..3990
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3841..3976
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3903..3922
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4039..5064
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4039..5064
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5194..5315
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5231..5250)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5285..5304
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5368..6159,6160..6228))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5991..6010
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6229..6320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6362..6381
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6387..6406
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggagagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1501 ggtcattagt tcatagccca tatatggagt tccgcgttac atagcatcgt acgcgtacgt
     1561 gtttggggaa agggacggag ccaagagcca ccatggtgag caagggcgag gagctgttca
     1621 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1681 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1741 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1801 agtgcttcag ccgttacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1861 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1921 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1981 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2041 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     2101 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2161 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2221 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2281 tcactctcgg catggacgag ctgtacaagg gaagcggaga gggcagagga agtctgctaa
     2341 catgcggtga cgtcgaggag aatcctggac ctatgaccga gtacaagccc acggtgcgcc
     2401 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     2461 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     2521 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     2581 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     2641 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     2701 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     2761 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     2821 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     2881 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     2941 cctggtgcat gacccgcaag cccggtgccg gaagcggagc tactaacttc agcctgctga
     3001 agcaggctgg cgacgtggag gagaaccctg gacctatgag gggttcacat catcaccacc
     3061 atcatgacta caaagacgat gacgacaagg actacaaaga cgatgacgac aaggactaca
     3121 aagacgatga cgacaagggc ggcaaggaga agagtgcttg tcctaaagat ccagccaaac
     3181 ctccggccaa ggcacaagtt gtgggatggc caccggtgag atcataccgg aagaacgtga
     3241 tggtttcctg ccaaaaatca agcggtggcc cggaggcggc ggcgttcgtg aaggtatcaa
     3301 tggacggagc accgtacttg aggaaaatcg atttgaggat gtataaagcg gcggccgacg
     3361 gcgacgacca aacacgtacg cgtacgatgc tctagaatgc tgatgggcta gcaaaatcag
     3421 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3481 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcac
     3541 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3601 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     3661 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3721 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     3781 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3841 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3901 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3961 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4021 ggatctgatc agcacgtgat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt
     4081 ctgatcgaaa agttcgacag cgtttccgac ctgatgcagc tctcggaggg cgaagaatct
     4141 cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc
     4201 gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt
     4261 ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt
     4321 gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
     4381 gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc
     4441 ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt
     4501 gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc
     4561 gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc
     4621 gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc
     4681 attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc
     4741 tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg
     4801 gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc
     4861 tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac
     4921 gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg
     4981 gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc
     5041 actcgtccga gggcaaagga atagcacgtg ctacgagatt tcgattccac cgccgccttc
     5101 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5161 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5221 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5281 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5341 agctagagct tggcgtaatc attgtcatta ccaatgctta atcagtgagg cacctatctc
     5401 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5461 gatacgggag ggcttaccat ctggccccag cgctgcgatg ataccgcgag aaccacgctc
     5521 accggctccg gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5581 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5641 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc
     5701 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5761 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5821 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5881 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5941 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6001 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6061 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6121 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6181 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatat tcttcctttt
     6241 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6301 tatttagaaa aataaacaaa taggggtcag tgttacaacc aattaaccaa ttctgaacat
     6361 tatcgcgagc ccatttatac ctgaatatgg ctcataacac cccttg
//
