LOCUS       sequence-319711-        8577 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Overexpresses KCNMB3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8577)
  TITLE     These plasmids allow lentivirus-mediated overexpression of 
            understudied V5 tagged ion channels in mammalian cell lines.
REFERENCE   2  (bases 1 to 8577)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: IDG_KCNMB3_OE_1
FEATURES             Location/Qualifiers
     source          1..8577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        88..391
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        392..595
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     546..566
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    618..642
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1498..1522)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1524..1565
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1607..2195
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1660..1680)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2078..2089)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2267..2500
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2578..2699
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2615..2634)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2669..2688
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2739..2874
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2894..2913)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2895..2913)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2923..2940)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2923..2939)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2932..2954)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3081..3536
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3168..3187)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3378..3399
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3562..3666
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3667..3735,3736..4527)
                     /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(3885..3904)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4698..5286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5187..5206
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5440..5457
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5574..5595
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5610..5615,5616..5633,5634..5640)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5648..5664
                     /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     5653..5675
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5672..5688
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5672..5688
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5707..5727
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5709..5727
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5755..5981
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5982..6162
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6209..6334
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6827..7060
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7245..7289
                     /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        7464..7964
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7883..7901
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7976..8371
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8431..8548
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 actttggcgc cggctcgagg gggcccgggg ttattaatag taatcaatta cggggtcatt
       61 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      121 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      181 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      241 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      301 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      361 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      421 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      481 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      541 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
      601 gctaactgtc gggatcaaca agtttgtaca aaaaagcaga agggccgtca aggcccacca
      661 tgttccccct tctttatgag ctcactgcag tatctccttc tccctttccc caaaggacag
      721 cctttcctgc ctcagggaag aagagagaga cagactacag tgatggagac ccactagatg
      781 tgcacaagag gctgccatcc agtgctggag aggaccgagc cgtgatgctg gggtttgcca
      841 tgatgggctt ctcagtccta atgttcttct tgctcggaac aaccattcta aagcctttta
      901 tgctcagcat tcagagagaa gaatcgacct gcactgccat ccacacagat atcatggacg
      961 actggctgga ctgtgccttc acctgtggtg tgcactgcca cggtcagggg aagtacccgt
     1021 gtcttcaggt gtttgtgaac ctcagccatc caggtcagaa agctctccta cattataatg
     1081 aagaggctgt ccagataaat cccaagtgct tttacacacc taagtgccac caagatagaa
     1141 atgatttgct caacagtgct ctggacataa aagaattctt cgatcacaaa aatggaaccc
     1201 ccttttcatg cttctacagt ccagccagcc aatctgaaga tgtcattctt ataaaaaagt
     1261 atgaccaaat ggctatcttc cactgtttat tttggccttc actgactctg ctaggtggtg
     1321 ccctgattgt tggcatggtg agattaacac aacacctgtc cttactgtgt gaaaaatata
     1381 gcactgtagt cagagatgag gtaggtggaa aagtacctta tatagaacag catcagttca
     1441 aactgtgcat tatgaggagg agcaaaggaa gagcagagaa atcttcaggc ctcatgggcc
     1501 cagctttctt gtacaaagtg gttggtaagc ctatccctaa ccctctcctc ggtctcgatt
     1561 ctacgtagta atgagctagc gctaaccggt ggcgcgttaa gtcgacaatc aacctctgga
     1621 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     1681 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     1741 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     1801 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     1861 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     1921 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     1981 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     2041 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     2101 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     2161 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     2221 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     2281 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     2341 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     2401 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     2461 tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt
     2521 catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac
     2581 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2641 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     2701 catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2761 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2821 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga
     2881 cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     2941 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     3001 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     3061 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3121 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3181 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3241 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3301 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3361 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3421 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3481 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     3541 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     3601 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     3661 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3721 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3781 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3841 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3901 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3961 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4021 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     4081 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     4141 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     4201 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     4261 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     4321 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     4381 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     4441 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     4501 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4561 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4621 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4681 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4741 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4801 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     4861 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4921 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4981 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5041 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5101 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5161 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5221 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5281 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5341 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     5401 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     5461 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5521 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5581 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5641 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     5701 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
     5761 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
     5821 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
     5881 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
     5941 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
     6001 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     6061 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     6121 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     6181 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     6241 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     6301 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     6361 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     6421 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     6481 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     6541 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     6601 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     6661 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     6721 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     6781 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     6841 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     6901 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     6961 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     7021 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     7081 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     7141 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     7201 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     7261 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     7321 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     7381 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     7441 acctcccaac cccgagggga cccttgcgcc ttttccaagg cagccctggg tttgcgcagg
     7501 gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct gggtctcgca
     7561 cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc
     7621 cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc
     7681 ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga cagcgccagg
     7741 gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc
     7801 gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt
     7861 gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag cgcacgtcgg
     7921 cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggta ccaccatggc
     7981 caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag
     8041 catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat
     8101 cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct
     8161 gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga
     8221 gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc
     8281 tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga
     8341 attgctgccc tctggttatg tgtgggaggg cctgcagctg cagtagtaag aattctagat
     8401 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     8461 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     8521 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatcc
//
