LOCUS       sequence-306870-        7119 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression and purification of the truncated TMC2 protein 
            from Melopsittacus undulates. The protease cleavage site was 
            replaced with the HRV 3C site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7119)
  AUTHORS   Jia Y, Zhao Y, Kusakizako T, Wang Y, Pan C, Zhang Y, Nureki O, 
            Hattori M, Yan Z
  TITLE     TMC1 and TMC2 Proteins Are Pore-Forming Subunits of Mechanosensitive
            Ion Channels.
  JOURNAL   Neuron. 2020 Jan 22;105(2):310-321.e3. doi: 
            10.1016/j.neuron.2019.10.017. Epub 2019 Nov 21.
  PUBMED    31761710
REFERENCE   2  (bases 1 to 7119)
  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"; doi: 
            "10.1016/j.neuron.2019.10.017"; journalName: "Neuron"; date: 
            "2020-01-22- 22"; volume: "105"; issue: "2"; pages: "310-321.e3"
COMMENT     Sequence Label: GFP-MuTMC2truncated
FEATURES             Location/Qualifiers
     source          1..7119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /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     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /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
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     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"
     regulatory      4038..4047
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4053..4076
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             join(4083..4088,4089..4112,4113..4148,4149..4172)
                     /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="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             4914..4937
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(5089..5100)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6609..6743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6646..6665)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6700..6719
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  6772..6937
                     /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 cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc 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 acttgggcag 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 cggatccgcc accatgggat ctcatcatca tcatcatcat catcatgcag
     4081 caagtgcctg gagtcacccc caatttgaaa aaggcggcgg ctccggcgga ggatcgggcg
     4141 ggagtgcgtg gtcgcacccc caatttgaaa aagtctcaaa aggagaagaa ttgtttacgg
     4201 gagttgttcc aatccttgtt gaacttgatg gagatgttaa cggacataaa ttctctgttt
     4261 ctggagaagg agaaggagat gcaacatacg gaaagctaac gcttaaattc atctgcacaa
     4321 caggaaagct cccggtccct tggccaacac ttgttacaac acttacatac ggagttcagt
     4381 gcttctctcg atacccagat catatgaaac agcatgattt cttcaaatct gcaatgccag
     4441 aaggatacgt tcaggaacga acaatcttct tcaaagatga tggaaactac aagacccgtg
     4501 cagaagttaa attcgaagga gatacacttg ttaaccgaat cgaacttaaa ggaatcgatt
     4561 tcaaagaaga tggaaacatc cttggacata aacttgaata caactacaac tctcataacg
     4621 tttacatcat ggcagataaa cagaagaatg gcatcaaagt taacttcaag attcgtcata
     4681 acatcgaaga tggatctgtt cagcttgcag atcattacca gcagaacaca ccaatcggag
     4741 atggaccagt tcttcttcca gataaccatt acctttctac acagtctaaa ctttctaaag
     4801 atccaaacga gaagagggac catatggttc ttcttgagtt cgttacagca gcaggaatca
     4861 cacttggaat ggatgaactt tacaaatctg gacttcgatc cgcagcagca ggacttgaag
     4921 ttctatttca aggaccagaa ttcctttacg catacaagat gatgatggca aagaagtggg
     4981 taaagtttaa gcgagatttc gataacttca agacccaatg catcccatgg gaaatgaaga
     5041 taaaggaagt tgaatctcat ttcggatctt ctgttgcatc ttacttcatc ttccttcgat
     5101 ggatgtacgg agttaacctt gttctatttg gtcttatctt cggacttgta attatacctg
     5161 agagatcttc taaactttct cagatgtctt ctggaacatg gctttacttc gaacaggaag
     5221 catggcttga tgatcttcag cgaatggcac gaaacgcaaa cgaatctaca gcagatggag
     5281 atgatgataa cttcatcttc tcttggaaga tgtttacatc ttgggattac cttatcggaa
     5341 acccagaaac agcagataac aaattcgcat ctatcacaac atctttcaaa gaatctatcg
     5401 ttgatgaaca ggaatctaac aaagatgaga atatacatct tcgacgattc cttcgagttc
     5461 ttgcaaacgt tcttatcatc tgctgccttt gcggatctgg ataccttatc tacttcgttg
     5521 ttaaacgatc tcagacattc tctaagatgc agaatgtcgg atggtacgaa cgaaacgaag
     5581 ttgaaatcgt tatgtctctt cttggaatgt tctgcccacc attatttgag acaatcgcag
     5641 cacttgagaa ttatcatcca cgaatcggac ttaaatggca gcttggacga atcttcgcac
     5701 tgttcctagg taacctttac acattccttc ttgcacttat ggatgatgtt aacgagaagc
     5761 tcgcggaaga agatgttgtt aagaatatta cacattggac acttcttgga catcataact
     5821 cttctatggg aaatagttcg agcgctactc caccgcctct tgatccagca gatgttccac
     5881 gaggaccatg ctgggaaaca acagttggaa tcgagttcgt tcgacttaca gtttctgata
     5941 tccttgttac atacatcaca atccttgttg gagatttcat ccgagcatgc ttcgttcgat
     6001 tcgttaacta ctgctggtgc tgggatcttg aagcaggatt cccatcttac gcagagttcg
     6061 atatctctgg aaacgttctt ggacttgtat ttaatcaggg aatgatctgg atgggatctt
     6121 tctacgcacc aggacttgtt ggaatcaacg ttcttcgact tcttacatct atgtacttcc
     6181 agtgctgggc agttatgtct tctaacgttc cacatgaacg agtgtttaag gcatctaaat
     6241 ctaacaactt ctacatggga cttcttcttc ttatcctttt cctttctctt cttccagttg
     6301 catacacaat catgtctctt ccaccatctt tcgattgcgg accattctct ggaaagaagc
     6361 gtatgtacga agttatccag gaaacaatcg aacttgattt cccactgttt gtcgcaaaga
     6421 tcttctctta cgttgcaaac ccaggattga taatacccgc tattcttctt atggtttaaa
     6481 agcttgtcga gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac
     6541 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     6601 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     6661 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     6721 atgtatctta tcatgtctgg atctgatcac tgcttgagcc taggagatcc gaaccagata
     6781 agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct
     6841 acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc
     6901 cacccctccc agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta
     6961 tgtttttaat caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt
     7021 tctctgtcac agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac
     7081 tgaatatcaa cgcttatttg cagcctgaat ggcgaatgg
//
