LOCUS       sequence-318724-        8274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Overexpresses KCNK4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8274)
  TITLE     These plasmids allow lentivirus-mediated overexpression of 
            understudied V5 tagged ion channels in mammalian cell lines.
REFERENCE   2  (bases 1 to 8274)
  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_KCNK4_OE_1
FEATURES             Location/Qualifiers
     source          1..8274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..308
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        309..512
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     463..483
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    535..559
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1112..1136)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1138..1179
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1221..1809
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1274..1294)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1692..1703)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1881..2114
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2192..2313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2229..2248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2283..2302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2353..2488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2508..2527)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2509..2527)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2537..2554)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2537..2553)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2546..2568)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2695..3150
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2782..2801)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2992..3013
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3176..3280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3281..3349,3350..4141)
                     /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(3499..3518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4312..4900
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4801..4820
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5054..5071
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5188..5209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5224..5229,5230..5247,5248..5254)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5262..5278
                     /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     5267..5289
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5286..5302
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5286..5302
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5321..5341
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5323..5341
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5369..5595
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5596..5776
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5823..5948
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6441..6674
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6859..6903
                     /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        7078..7578
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7497..7515
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7590..7985
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8045..8162
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      301 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      361 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      421 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      481 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactgtcggg atcaacaagt
      541 ttgtacaaaa aagttggcat gacaacagct ccccaggagc cccccgcccg gcccctccag
      601 gcgggcagtg gagctggccc ggcgcctggg cgcgccatgc gcagcaccac gctcctggcc
      661 ctgctggcgc tggtcttgct ttacttggtg tctggtgccc tggtgttccg ggccctggag
      721 cagccccacg agcagcaggc ccagagggag ctgggggagg tccgagagaa gttcctgagg
      781 gcccatccgt gtgtgagcga ccaggagctg ggcctcctca tcaaggaggt ggctgatgcc
      841 ctgggagggg gtgcggaccc agaaaccaac tcgaccagca acagcagcca ctcagcctgg
      901 gacctgggca gcgccttctt tttctcaggg accatcatca ccaccatcgg ctatggcaat
      961 gtggccctgc gcacagatgc cgggcgcctc ttctgcatct tttatgcgct ggtggggatt
     1021 ccgctgtttg ggatcctact ggcaggggtc ggggaccggc tgggctcctc cctgcgccat
     1081 ggcatcggtc acattgaagc catcttcttg tgcccaactt tcttgtacaa agtggttggt
     1141 aagcctatcc ctaaccctct cctcggtctc gattctacgt agtaatgagc tagcgctaac
     1201 cggtggcgcg ttaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg
     1261 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     1321 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     1381 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     1441 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     1501 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     1561 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     1621 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     1681 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     1741 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     1801 cctccccgcg tcgactttaa gaccaatgac ttacaaggca gctgtagatc ttagccactt
     1861 tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac aagatctgct
     1921 ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     1981 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     2041 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     2101 gaaaatctct agcagtacgt atagtagttc atgtcatctt attattcagt atttataact
     2161 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
     2221 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2281 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
     2341 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     2401 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     2461 aagtagtgag gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt
     2521 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
     2581 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
     2641 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
     2701 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2761 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2821 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2881 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2941 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3001 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3061 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3121 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3181 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3241 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3301 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3361 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3421 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3481 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3541 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3601 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3661 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3721 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3781 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3841 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3901 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3961 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4021 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4081 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4141 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     4201 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     4261 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4321 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4381 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4441 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4501 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4561 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4621 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4681 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4741 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4801 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4861 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4921 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4981 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     5041 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     5101 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5161 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     5221 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5281 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
     5341 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
     5401 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
     5461 cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg
     5521 acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta
     5581 gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     5641 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     5701 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     5761 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
     5821 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
     5881 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     5941 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     6001 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     6061 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     6121 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     6181 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     6241 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     6301 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     6361 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     6421 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     6481 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     6541 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     6601 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     6661 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     6721 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     6781 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     6841 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     6901 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     6961 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     7021 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccttg
     7081 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
     7141 gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc
     7201 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
     7261 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt
     7321 ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga
     7381 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg
     7441 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt
     7501 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc
     7561 accgacctct ctccccaggg ggtaccacca tggccaagcc tttgtctcaa gaagaatcca
     7621 ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg
     7681 tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt
     7741 ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg
     7801 gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg
     7861 gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca
     7921 gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg
     7981 agggcctgca gctgcagtag taagaattct agatcttgag acaaatggca gtattcatcc
     8041 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     8101 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     8161 ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag ggggcccggg
     8221 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagt
//
