LOCUS       sequence-310813-        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"
     rep_origin      2..457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     299..320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        484..588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(589..657,658..1449)
                     /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(807..826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1620..2208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2109..2128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2362..2379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3524..3529,3530..3546,3547..3552))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        3904..3995
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     3920..3937
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        4175..4554
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4555..4758
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4709..4729
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4755..4779
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          4894..5026
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     5009..5028
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             6198..6221
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             6231..6269
                     /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(6279..6302,6303..6338,6339..6362)
                     /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             6369..6392
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     misc_feature    6423..7011
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6476..6496)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6894..6905)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7162..7296
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7199..7218)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7253..7272
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7325..7490
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      301 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      361 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      421 aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat
      481 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
      541 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
      601 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
      661 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
      721 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
      781 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
      841 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
      901 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
      961 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     1021 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     1081 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     1141 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     1201 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     1261 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     1321 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     1381 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     1441 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     1501 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1741 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1981 tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg
     2041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2281 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2341 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     2401 cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3361 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     3421 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     3481 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     3541 ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac
     3601 cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc
     3661 ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
     3721 cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt
     3781 ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt
     3841 gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa
     3901 tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt
     3961 ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca
     4021 ccatcgggcg cggatcctca atattggcca ttagccatat tattcattgg ttatatagca
     4081 taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa tatgtacatt
     4141 tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac tagttattaa
     4201 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     4261 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     4321 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     4381 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc
     4441 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     4501 cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     4561 cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     4621 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     4681 aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag
     4741 gtctatataa gcagagctcg tttagtgaac cgtcagatca ctagaagctt tattgcggta
     4801 gtttatcaca gttaaattgc taacgcagtc agtgcttctg acacaacagt ctcgaactta
     4861 agctgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac aagacaggtt
     4921 taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg tttctgatag
     4981 gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt ccactcccag
     5041 ttcaattaca gctcttaagg gaacccggtc cgaagcgcgc ggaattcgcc accatgagca
     5101 gagagtttga cttcttcacc tatgagaccc ccaaggtgat tgtggtgaag agctggacca
     5161 ttggcatcat caacagagtg gtgcagctgc tgatcatcag ctactttgtg ggctgggtgt
     5221 tcctgcatga gaaggcctac caagtgagag acacagccat tgagagctct gtggtgacca
     5281 aggtgaaggg ctctggcctg tatgccaaca gagtgatgga tgtgtctgac tatgtgaccc
     5341 ccccccaagg cacctctgtg tttgtgatca tcaccaagat gattgtgaca gagaatcaga
     5401 tgcaaggctt ctgccctgag tctgaggaga agtacagatg tgtgtctgac tctcagtgtg
     5461 gccctgagag actgcctggg gggggcatcc tgactggcag atgtgtgaac tacagctctg
     5521 tgctgagaac ctgtgagatc caaggctggt gccccacaga ggtggacaca gtggagaccc
     5581 ccatcatgat ggaggctgag aacttcacca tcttcatcaa gaacagcatc agattccccc
     5641 tgttcaactt tgagaagggc aacctgctgc ccaacctgac agctagagac atgaagacct
     5701 gcagattcca ccctgacaag gaccccttct gccccatcct gagagtgggg gatgtggtga
     5761 agtttgctgg ccaagacttt gccaagctgg ctagaactgg gggggtgctg ggcatcaaga
     5821 ttggctgggt gtgtgacctg gacaaggcct gggatcagtg catccccaag tacagcttca
     5881 caagactgga ctctgtgtct gagaagagct ctgtgagccc tggctacaac ttcagatttg
     5941 ccaagtacta caagatggag aatggctctg agtacagaac cctgctgaag gcctttggca
     6001 tcagatttga tgtgctggtg tatggcaatg ctggcaagtt caacatcatc cccaccatca
     6061 tcagctctgt ggctgccttc acctctgtgg gggtgggcac agtgctgtgt gacatcatcc
     6121 tgctgaactt cctgaagggg gctgatcagt acaaggccaa gaagtttgaa gaggtgaatg
     6181 agaccgtcga cggcagcctg gaggtgctgt tccagggccc cagcggcggg agccgtctcg
     6241 aggaggagct gagaagaaga ctgaccgaag gcggcagctg gagccatccc cagttcgaaa
     6301 aaggcggggg ctccggtggt ggaagcgggg gatctgcgtg gagccacccg cagttcgaga
     6361 agggcagcca ccaccaccac caccaccacc actaatctag agcctgcagt ctcgacaagc
     6421 ttaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     6481 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     6541 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     6601 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     6661 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     6721 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     6781 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     6841 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     6901 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     6961 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cctgctcgag
     7021 gcatgcggta ccaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt
     7081 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     7141 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     7201 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     7261 tccaaactca tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctaggaga
     7321 tccgaaccag ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt
     7381 agcttacgac gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta
     7441 aaaactccat ttccacccct cccagttccc aactattttg tccgcccaca gcggggcatt
     7501 tttcttcctg ttatgttttt aatcaaacat cctgccaact ccatgtgaca aaccgtcatc
     7561 ttcggctact ttttctctgt cacagaatga aaatttttct gtcatctctt cgttattaat
     7621 gtttgtaatt gactgaatat caacgcttat ttgcagcctg aatggcgaat gg
//
