LOCUS       sequence-478961-        8254 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Overexpression of PCP-3xSNAP in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8254)
  AUTHORS   Yang LZ, Min YH, Liu YX, Gao BQ, Liu XQ, Huang Y, Wang H, Yang L, 
            Liu ZJ, Chen LL
  TITLE     CRISPR-array-mediated imaging of non-repetitive and multiplex 
            genomic loci in living cells.
  JOURNAL   Nat Methods. 2024 Sep;21(9):1646-1657. doi: 
            10.1038/s41592-024-02333-3. Epub 2024 Jul 4.
  PUBMED    38965442
REFERENCE   2  (bases 1 to 8254)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02333-3"; journalName: "Nat Methods"; date: 
            "2024-09"; volume: "21"; issue: "9"; pages: "1646-1657"
COMMENT     Sequence Label: pHAGE-EFS-PCP-3xSNAP-NLS
FEATURES             Location/Qualifiers
     source          1..8254
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2265..2476
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2522..2531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2933..3478
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             3497..4042
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             4061..4606
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             4613..4633
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    4644..5232
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4697..4717)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5115..5126)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5307..5540
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5659..5677)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5745..5849
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5850..5918,5919..6710)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6068..6087)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6881..7469
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7370..7389
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7623..7640
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7880..7901
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7916..7921,7922..7939,7940..7946)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7954..7970
                     /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)."
     primer_bind     7959..7981
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7978..7994
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7978..7994
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8044..8063
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttgacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagcgat
     2221 gctagctgat gcgggccctt cgagtggctc cggtgcccgt cagtgggcag agcgcacatc
     2281 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     2341 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     2401 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     2461 tgccgccaga acacaggtgt cgtgacgcgg ttccggttaa ggtgaacgcg tgcaggctgg
     2521 cgccaccatg ggttccaaaa ccatcgttct ttcggtcggc gaggctactc gcactctgac
     2581 tgagatccag tccaccgcag accgtcagat cttcgaagag aaggtcgggc ctctggtggg
     2641 tcggctgcgc ctcacggctt cgctccgtca aaacggagcc aagaccgcgt atcgcgtcaa
     2701 cctaaaactg gatcaggcgg acgtcgttga ttccggactt ccgaaagtgc gctacactca
     2761 ggtatggtcg cacgacgtga caatcgttgc gaatagcacc gaggcctcgc gcaaatcgtt
     2821 gtacgatttg accaagtccc tcgtcgcgac ctcgcaggtc gaagatcttg tcgtcaacct
     2881 tgtgccgctg ggccgtggtg gcggagggac tagtggaggt ggatccggaa gtatggacaa
     2941 agactgcgaa atgaagcgca ccaccctgga tagccctctg ggcaagctgg aactgtctgg
     3001 gtgcgaacag ggcctgcacc gtatcatctt cctgggcaaa ggaacatctg ccgccgacgc
     3061 cgtggaagtg cctgccccag ccgccgtgct gggcggacca gagccactga tgcaggccac
     3121 cgcctggctc aacgcctact ttcaccagcc tgaggccatc gaggagttcc ctgtgccagc
     3181 cctgcaccac ccagtgttcc agcaggagag ctttacccgc caggtgctgt ggaaactgct
     3241 gaaagtggtg aagttcggag aggtcatcag ctacagccac ctggccgccc tggccggcaa
     3301 tcccgccgcc accgccgccg tgaaaaccgc cctgagcgga aatcccgtgc ccattctgat
     3361 cccctgccac cgggtggtgc agggcgacct ggacgtgggg ggctacgagg gcgggctcgc
     3421 cgtgaaagag tggctgctgg cccacgaggg ccacagactg ggcaagcctg ggctgggttc
     3481 tggcggccgc tctggcatgg acaaagactg cgaaatgaag cgcaccaccc tggatagccc
     3541 tctgggcaag ctggaactgt ctgggtgcga acagggcctg caccgtatca tcttcctggg
     3601 caaaggaaca tctgccgccg acgccgtgga agtgcctgcc ccagccgccg tgctgggcgg
     3661 accagagcca ctgatgcagg ccaccgcctg gctcaacgcc tactttcacc agcctgaggc
     3721 catcgaggag ttccctgtgc cagccctgca ccacccagtg ttccagcagg agagctttac
     3781 ccgccaggtg ctgtggaaac tgctgaaagt ggtgaagttc ggagaggtca tcagctacag
     3841 ccacctggcc gccctggccg gcaatcccgc cgccaccgcc gccgtgaaaa ccgccctgag
     3901 cggaaatccc gtgcccattc tgatcccctg ccaccgggtg gtgcagggcg acctggacgt
     3961 ggggggctac gagggcgggc tcgccgtgaa agagtggctg ctggcccacg agggccacag
     4021 actgggcaag cctgggctgg gtggtagcat cgatggtagc atggacaaag actgcgaaat
     4081 gaagcgcacc accctggata gccctctggg caagctggaa ctgtctgggt gcgaacaggg
     4141 cctgcaccgt atcatcttcc tgggcaaagg aacatctgcc gccgacgccg tggaagtgcc
     4201 tgccccagcc gccgtgctgg gcggaccaga gccactgatg caggccaccg cctggctcaa
     4261 cgcctacttt caccagcctg aggccatcga ggagttccct gtgccagccc tgcaccaccc
     4321 agtgttccag caggagagct ttacccgcca ggtgctgtgg aaactgctga aagtggtgaa
     4381 gttcggagag gtcatcagct acagccacct ggccgccctg gccggcaatc ccgccgccac
     4441 cgccgccgtg aaaaccgccc tgagcggaaa tcccgtgccc attctgatcc cctgccaccg
     4501 ggtggtgcag ggcgacctgg acgtgggggg ctacgagggc gggctcgccg tgaaagagtg
     4561 gctgctggcc cacgagggcc acagactggg caagcctggg ctgggtctcg agccaaagaa
     4621 aaagcggaaa gtgtaatcta gataatcaac ctctggatta caaaatttgt gaaagattga
     4681 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     4741 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     4801 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     4861 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     4921 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     4981 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     5041 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     5101 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     5161 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     5221 ccgcctcccc gcctgagatc ctttaagacc aatgacttac aaggcagctg tagatcttag
     5281 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     5341 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     5401 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     5461 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     5521 agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact
     5581 tgcaaagaaa tgaatatcag agagtgagag gcccgggtta attaaggaaa gggctagatc
     5641 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5701 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5761 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5821 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5881 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5941 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6001 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6061 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     6121 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6181 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6241 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6301 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6361 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6421 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6481 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6541 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6601 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6661 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6721 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6781 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6841 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6901 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6961 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7021 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7081 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7141 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7201 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7261 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7321 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7381 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7441 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7501 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     7561 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     7621 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     7681 cgcgttggcc gattcattaa tgcagcaagc tcatggctga ctaatttttt ttatttatgc
     7741 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     7801 aggcctaggc ttttgcaaaa agctccccgt ggcacgacag gtttcccgac tggaaagcgg
     7861 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     7921 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     7981 gaaacagcta tgacatgatt acgaatttca caaataaagc atttttttca ctgcattcta
     8041 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc
     8101 aagctaacca aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc
     8161 tgtggtttca tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt
     8221 gtatttgtta aatatgtact acaaacttag tagt
//
