LOCUS       sequence-248880-        6202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides the repair template with PP2Ac microhomology on each end 
            for PP2Ac N-ter H3F tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6202)
  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 6202)
  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-HF
FEATURES             Location/Qualifiers
     source          1..6202
                     /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)."
     CDS             join(1595..1597,1598..1600,1601..2311)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1595..1597,1598..1600,1601..2311)
                     /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(1640..1661)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1901..1920)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2248..2269
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2321..2374
                     /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             2375..2971
                     /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(2375..2394)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2871..2891
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2981..3037
                     /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             3050..3067
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3068..3139
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(3217..3234)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3223..3334
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3452..3472)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3457..3786
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3637..3772
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3699..3718
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3835..4860
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3835..4860
                     /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    4990..5111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5027..5046)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5081..5100
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5164..5955,5956..6024))
                     /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     5787..5806
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6025..6116)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6158..6177
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6183..6202
                     /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 atagcaccgt acgcgtacgt
     1561 gtttggggcg ggtggcatca tggacgccgg atccatggtg agcaagggcg aggagctgtt
     1621 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     1681 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     1741 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     1801 gcagtgcttc agccgttacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     1861 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     1921 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     1981 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     2041 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     2101 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     2161 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     2221 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     2281 gatcactctc ggcatggacg agctgtacaa gggaagcgga gagggcagag gaagtctgct
     2341 aacatgcggt gacgtcgagg agaatcctgg acctatgacc gagtacaagc ccacggtgcg
     2401 cctcgccacc cgcgacgacg tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga
     2461 ctaccccgcc acgcgccaca ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct
     2521 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     2581 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     2641 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     2701 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     2761 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     2821 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     2881 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     2941 cacctggtgc atgacccgca agcccggtgc cggaagcgga gctactaact tcagcctgct
     3001 gaagcaggct ggcgacgtgg aggagaaccc tggacctatg aggggttcac atcatcacca
     3061 ccatcatgac tacaaagacg atgacgacaa ggactacaaa gacgatgacg acaaggacta
     3121 caaagacgat gacgacaagg acgagaaggt gttcaccaag gagccaaaca cgtacgcgta
     3181 cgatgctcta gaatgctgat gggctagcaa aatcagcctc gactgtgcct tctagttgcc
     3241 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3301 ctgtcctttc ctaataaaat gaggaaattg catcacaaca ctcaacccta tctcggtcta
     3361 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3421 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3481 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3541 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3601 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3661 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3721 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3781 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgatgaaa
     3841 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtt
     3901 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3961 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     4021 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     4081 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     4141 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     4201 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     4261 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     4321 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     4381 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4441 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4501 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4561 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4621 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4681 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4741 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4801 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     4861 cacgtgctac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4921 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4981 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5041 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5101 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcattg
     5161 tcattaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5221 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5281 ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat
     5341 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5401 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5461 caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5521 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5581 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5641 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5701 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5761 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5821 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5881 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5941 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6001 gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca
     6061 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6121 ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga
     6181 atatggctca taacacccct tg
//
