LOCUS       sequence-319632-        8604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpresses TMC4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8604)
  TITLE     These plasmids allow lentivirus-mediated overexpression of 
            understudied V5 tagged ion channels in mammalian cell lines.
REFERENCE   2  (bases 1 to 8604)
  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"
COMMENT     Sequence Label: IDG_TMC4_OE_1
FEATURES             Location/Qualifiers
     source          1..8604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    531..555
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1438..1462)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1464..1505
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1547..2135
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1600..1620)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2018..2029)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2207..2440
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2518..2639
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2555..2574)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2609..2628
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2679..2814
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2834..2853)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2835..2853)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2863..2880)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2863..2879)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2872..2894)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3021..3476
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3108..3127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3318..3339
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3502..3606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3607..3675,3676..4467)
                     /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(3825..3844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4638..5226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5127..5146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5380..5397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5514..5535
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5550..5555,5556..5573,5574..5580)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5588..5604
                     /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     5593..5615
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5612..5628
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5612..5628
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5647..5667
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5649..5667
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5695..5921
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5922..6102
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6149..6274
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6767..7000
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7185..7229
                     /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"
     promoter        7404..7904
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7823..7841
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7916..8311
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8371..8488
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctct ctggctaact gtcgggatca acaagtttgt
      541 acaaaaaagt tggcatgaag ggtgggggcg gtcagaggga tgttggcgct gacaggaccg
      601 tgtttcttcg cctcgcctcc ctggtggtcc tgctcttctc tctctggaat cagatcactt
      661 gtgggggcga ctccgaggct gaggactgca aaacctgtgg ctacaattac aaacaacttc
      721 cgtgctggga gactgtcctg ggccaggaaa tgtacaaact tctgctcttt gatctgctga
      781 ctgtcttggc agtcgcgctg ctcatccagt ttcctagaaa gctcctctgt ggcctctgtc
      841 ctggggcgct gggtcgtctg gcggggaccc aggagttcca ggtgcccgac gaggtgctgg
      901 ggctcatcta cgcgcagacg gtggtctggg tggggagttt tttctgccct ttactgcccc
      961 tgcttaacac ggtcaagttc ctgctgcttt tctacctgaa gaagcttacc ctcttctcca
     1021 cctgctcccc ggctgcccgc accttccggg cctccgcggc gaatttcttt ttccccttgg
     1081 tccttctcct gggtctggcc atctccagcg ttcccctgct ttacagcatc ttcctgatcc
     1141 cgccttctaa gctttgtggt ccattccggg ggcagtcgtc catctgggcc cagatccctg
     1201 agtctatttc cagcctccct gagaccaccc agaatttcct cttcttcctg gggacccagg
     1261 cttttgctgt gccccttctg ctgatctcca gcatcctgat ggcgtacact gtggctctgg
     1321 ctaactccta cggacgcctc atctctgagc tcaaacgtca gagacagacg gaggcgcaga
     1381 ataaagtctt cctggcacgg cgcgctgtgg cgctgacctc caccaaaccg gctctttgcc
     1441 caactttctt gtacaaagtg gttggtaagc ctatccctaa ccctctcctc ggtctcgatt
     1501 ctacgtagta atgagctagc gctaaccggt ggcgcgttaa gtcgacaatc aacctctgga
     1561 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     1621 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     1681 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     1741 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     1801 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     1861 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     1921 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     1981 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     2041 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     2101 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     2161 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     2221 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     2281 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     2341 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     2401 tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt
     2461 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     2521 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2581 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     2641 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2701 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2761 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     2821 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     2881 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     2941 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     3001 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3061 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3121 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3181 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3241 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3301 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3361 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3421 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     3481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3541 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3601 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3661 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3721 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3781 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3841 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3901 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3961 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4021 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4081 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4141 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4201 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4261 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4321 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4381 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4441 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4501 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4561 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4621 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4681 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4741 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4801 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4861 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4921 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4981 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5041 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5101 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5161 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5221 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5281 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5341 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5401 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5461 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5521 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5581 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     5641 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     5701 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     5761 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     5821 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     5881 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     5941 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     6001 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6061 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     6121 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     6181 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     6241 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     6301 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     6361 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     6421 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     6481 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     6541 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     6601 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     6661 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     6721 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     6781 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     6841 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     6901 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     6961 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     7021 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     7081 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     7141 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     7201 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     7261 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     7321 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     7381 acctcccaac cccgagggga cccttgcgcc ttttccaagg cagccctggg tttgcgcagg
     7441 gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca
     7501 cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc
     7561 cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc
     7621 ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg
     7681 gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc
     7741 gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt
     7801 gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg
     7861 cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggta ccaccatggc
     7921 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     7981 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     8041 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     8101 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     8161 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     8221 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     8281 attgctgccc tctggttatg tgtgggaggg cctgcagctg cagtagtaag aattctagat
     8341 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     8401 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     8461 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
     8521 ttggcgccgg ctcgaggggg cccggggtta ttaatagtaa tcaattacgg ggtcattagt
     8581 tcatagccca tatatggagt tccg
//
