LOCUS       sequence-248877-        6441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Provides the repair template with PP2Ac microhomology on each end 
            for PP2Ac N-ter HBTH tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6441)
  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 6441)
  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-HBTH
FEATURES             Location/Qualifiers
     source          1..6441
                     /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(1587..1589,1590..1592,1593..2303)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1587..1589,1590..1592,1593..2303)
                     /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(1632..1653)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1893..1912)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2240..2261
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2313..2366
                     /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             2367..2963
                     /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(2367..2386)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2863..2883
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2973..3029
                     /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             3033..3050
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3303..3323
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3345..3362
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3456..3473)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3462..3573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3691..3711)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3696..4025
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3876..4011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3938..3957
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4074..5099
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4074..5099
                     /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    5229..5350
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5266..5285)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5320..5339
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5403..6194,6195..6263))
                     /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     6026..6045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6264..6355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6397..6416
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6422..6441
                     /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 gtttggtccc ggcctccctc ctccatatgg tgagcaaggg cgaggagctg ttcaccgggg
     1621 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1681 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1741 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1801 tcagccgtta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1861 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1921 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1981 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2041 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2101 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2161 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2221 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2281 tcggcatgga cgagctgtac aagggaagcg gagagggcag aggaagtctg ctaacatgcg
     2341 gtgacgtcga ggagaaccct ggacctatga ccgagtacaa gcccacggtg cgcctcgcca
     2401 cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg
     2461 ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac
     2521 tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg
     2581 cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg
     2641 gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc
     2701 tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc
     2761 ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg
     2821 agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg
     2881 agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt
     2941 gcatgacccg caagcccggt gccggaagcg gagctactaa cttcagcctg ctgaagcagg
     3001 ctggcgacgt ggaggagaac cctggaccta tgcatcatca ccaccatcat gctggaaagg
     3061 ccggtgaagg tgaaatccct gcccctcttg ctggtaccgt ttctaagata ctggtaaaag
     3121 aaggcgacac tgttaaagct ggtcaaacag ttctggtgct ggaggctatg aaaatggaga
     3181 cagaaattaa cgctcctact gacggaaaag ttgaaaaggt gttagttaag gaaagagatg
     3241 ctgttcaagg tggtcaaggt ctaatcaaga tcggcgacta cgatataccc acaaccgcca
     3301 gcgagaatct gtactttcaa ggagagttag ggatgagggg ttcacatcat caccaccatc
     3361 atatggaaga aggaggtaga gacaaggcgc cggtgcagcc ccccaaacac gtacgcgtac
     3421 gatgctctag aatgctgatg ggctagcaaa atcagcctcg actgtgcctt ctagttgcca
     3481 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3541 tgtcctttcc taataaaatg aggaaattgc atcacaacac tcaaccctat ctcggtctat
     3601 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3661 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3901 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3961 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4021 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgatgaaaa
     4081 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgttt
     4141 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     4201 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     4261 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat
     4321 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     4381 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     4441 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     4501 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     4561 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     4621 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     4681 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     4741 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     4801 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     4861 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     4921 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     4981 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     5041 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     5101 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5161 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5221 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5281 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5341 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcattgt
     5401 cattaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5461 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5521 cccagcgctg cgatgatacc gcgagaacca cgctcaccgg ctccggattt atcagcaata
     5581 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5641 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5701 aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5761 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5821 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5881 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5941 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6001 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6061 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6121 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6181 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6241 acacggaaat gttgaatact catattcttc ctttttcaat attattgaag catttatcag
     6301 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6361 gtcagtgtta caaccaatta accaattctg aacattatcg cgagcccatt tatacctgaa
     6421 tatggctcat aacacccctt g
//
