LOCUS       sequence-320663-        7672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression of human P2X3 with ALFA tag at C-terminus in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7672)
  AUTHORS   Jin F, Shen C, Wang Y, Wang M, Sun M, Hattori M
  TITLE     Fluorescence-detection size-exclusion chromatography utilizing 
            nanobody technology for expression screening of membrane proteins.
  JOURNAL   Commun Biol. 2021 Mar 19;4(1):366. doi: 10.1038/s42003-021-01891-y.
  PUBMED    33742097
REFERENCE   2  (bases 1 to 7672)
  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: "Commun 
            Biol."; date: "2021-03-19"; volume: "4(1):366. doi"; pages: " 
            10.1038/s42003-021-01891-y"
COMMENT     Sequence Label: hP2X3-cALFA
FEATURES             Location/Qualifiers
     source          1..7672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     17..34
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        272..651
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        652..855
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     806..826
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     852..876
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          991..1123
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1106..1125
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             2295..2318
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2328..2366
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             join(2376..2399,2400..2435,2436..2459)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             2466..2489
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     misc_feature    2520..3108
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2573..2593)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2991..3002)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3259..3393
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3296..3315)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3350..3369
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  3422..3587
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      3771..4225
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3858..3877)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4067..4088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4251..4355
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4356..4424,4425..5216)
                     /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     complement(4574..4593)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5387..5975
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5876..5895
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6129..6146
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  6280..6504
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(6571..7104)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(7293..7298,7299..7315,7316..7321))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc
       61 gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca
      121 tcgggcgcgg atcctcaata ttggccatta gccatattat tcattggtta tatagcataa
      181 atcaatattg gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat
      241 attggctcat gtccaatatg accgccatgt tggcattgat tattgactag ttattaatag
      301 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      361 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      421 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      481 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct
      541 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg
      601 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      661 ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      721 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      781 tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      841 tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt
      901 tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc
      961 tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa
     1021 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
     1081 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc
     1141 aattacagct cttaagggaa cccggtccga agcgcgcgga attcgccacc atgagcagag
     1201 agtttgactt cttcacctat gagaccccca aggtgattgt ggtgaagagc tggaccattg
     1261 gcatcatcaa cagagtggtg cagctgctga tcatcagcta ctttgtgggc tgggtgttcc
     1321 tgcatgagaa ggcctaccaa gtgagagaca cagccattga gagctctgtg gtgaccaagg
     1381 tgaagggctc tggcctgtat gccaacagag tgatggatgt gtctgactat gtgacccccc
     1441 cccaaggcac ctctgtgttt gtgatcatca ccaagatgat tgtgacagag aatcagatgc
     1501 aaggcttctg ccctgagtct gaggagaagt acagatgtgt gtctgactct cagtgtggcc
     1561 ctgagagact gcctgggggg ggcatcctga ctggcagatg tgtgaactac agctctgtgc
     1621 tgagaacctg tgagatccaa ggctggtgcc ccacagaggt ggacacagtg gagaccccca
     1681 tcatgatgga ggctgagaac ttcaccatct tcatcaagaa cagcatcaga ttccccctgt
     1741 tcaactttga gaagggcaac ctgctgccca acctgacagc tagagacatg aagacctgca
     1801 gattccaccc tgacaaggac cccttctgcc ccatcctgag agtgggggat gtggtgaagt
     1861 ttgctggcca agactttgcc aagctggcta gaactggggg ggtgctgggc atcaagattg
     1921 gctgggtgtg tgacctggac aaggcctggg atcagtgcat ccccaagtac agcttcacaa
     1981 gactggactc tgtgtctgag aagagctctg tgagccctgg ctacaacttc agatttgcca
     2041 agtactacaa gatggagaat ggctctgagt acagaaccct gctgaaggcc tttggcatca
     2101 gatttgatgt gctggtgtat ggcaatgctg gcaagttcaa catcatcccc accatcatca
     2161 gctctgtggc tgccttcacc tctgtggggg tgggcacagt gctgtgtgac atcatcctgc
     2221 tgaacttcct gaagggggct gatcagtaca aggccaagaa gtttgaagag gtgaatgaga
     2281 ccgtcgacgg cagcctggag gtgctgttcc agggccccag cggcgggagc cgtctcgagg
     2341 aggagctgag aagaagactg accgaaggcg gcagctggag ccatccccag ttcgaaaaag
     2401 gcgggggctc cggtggtgga agcgggggat ctgcgtggag ccacccgcag ttcgagaagg
     2461 gcagccacca ccaccaccac caccaccact aatctagagc ctgcagtctc gacaagctta
     2521 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2581 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2641 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     2701 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     2761 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     2821 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     2881 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg
     2941 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3001 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3061 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct gctcgaggca
     3121 tgcggtacca agcttgtcga gaagtactag aggatcataa tcagccatac cacatttgta
     3181 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3241 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3301 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3361 aaactcatca atgtatctta tcatgtctgg atctgatcac tgcttgagcc taggagatcc
     3421 gaaccagata agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc
     3481 ttacgacgct acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa
     3541 actccatttc cacccctccc agttcccaac tattttgtcc gcccacagcg gggcattttt
     3601 cttcctgtta tgtttttaat caaacatcct gccaactcca tgtgacaaac cgtcatcttc
     3661 ggctactttt tctctgtcac agaatgaaaa tttttctgtc atctcttcgt tattaatgtt
     3721 tgtaattgac tgaatatcaa cgcttatttg cagcctgaat ggcgaatggg acgcgccctg
     3781 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3841 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3901 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3961 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4021 atagacggtt tttcgccctt tgacgttgga gtccacgttc ttaatagtgg actcttgttc
     4081 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4141 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4201 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     4261 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     4321 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     4381 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     4441 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     4501 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     4561 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     4621 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     4681 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     4741 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     4801 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     4861 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     4921 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     4981 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5041 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5101 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5161 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     5221 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5281 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5341 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5401 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5461 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5521 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5581 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5641 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5701 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5761 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5821 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5881 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5941 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6001 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6061 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     6121 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     6181 ttacgcatct gtgcggtatt tcacaccgca tagaccagcc gcgtaacctg gcaaaatcgg
     6241 ttacggttga gtaataaatg gatgccctgc gtaagcgggt gtgggcggac aataaagtct
     6301 taaactgaac aaaatagatc taaactatga caataaagtc ttaaactaga cagaatagtt
     6361 gtaaactgaa atcagtccag ttatgctgtg aaaaagcata ctggactttt gttatggcta
     6421 aagcaaactc ttcattttct gaagtgcaaa ttgcccgtcg tattaaagag gggcgtggcc
     6481 aagggcatgg taaagactat attcgcggcg ttgtgacaat ttaccgaaca actccgcggc
     6541 cgggaagccg atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa
     6601 gtgcatcact tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc
     6661 gtaatctgct tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag
     6721 attgatgagc gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata
     6781 gatatagatc tcactacgcg gctgctcaaa cttgggcaga acgtaagccg cgagagcgcc
     6841 aacaaccgct tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt
     6901 cccgaggtaa tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg
     6961 accgaaaaga tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt
     7021 gcgaatgatg cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt
     7081 aacatcgttg ctgctgcgta acatcgttgc tgctccataa catcaaacat cgacccacgg
     7141 cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg tacaaaaaaa cagtcataac
     7201 aagccatgaa aaccgccact gcgccgttac caccgctgcg ttcggtcaag gttctggacc
     7261 agttgcgtga gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca
     7321 actgggttcg tgccttcatc cgtttccacg gtgtgcgtca cccggcaacc ttgggcagca
     7381 gcgaagtcga ggcatttctg tcctggctgg cgaacgagcg caaggtttcg gtctccacgc
     7441 atcgtcaggc attggcggcc ttgctgttct tctacggcaa ggtgctgtgc acggatctgc
     7501 cctggcttca ggagatcgga agacctcggc cgtcgcggcg cttgccggtg gtgctgaccc
     7561 cggatgaagt ggttcgcatc ctcggttttc tggaaggcga gcatcgtttg ttcgcccagg
     7621 actctagcta tagttctagt ggttggctac gtatactccg gaatattaat ag
//
