LOCUS       sequence-405493-        6355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding KCNE2 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6355)
  AUTHORS   Moyer AJ, Fernandez FX, Li Y, Klinedinst DK, Florea LD, Kazuki Y, 
            Oshimura M, Reeves RH
  TITLE     Overexpression screen of chromosome 21 genes reveals modulators of 
            Sonic hedgehog signaling relevant to Down syndrome.
  JOURNAL   Dis Model Mech. 2023 Apr 1;16(4):dmm049712. doi: 10.1242/dmm.049712.
            Epub 2023 Apr 13.
  PUBMED    36995257
REFERENCE   2  (bases 1 to 6355)
  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: "Dis Model 
            Mech."; date: "2023-04-1"; volume: "16(4):dmm049712. doi"; pages: " 
            10.1242/dmm.049712. Epub 2023 Apr 13"
COMMENT     Sequence Label: KCNE2_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6355
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     322..345
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     rep_origin      complement(395..823)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(505..526)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     717..736
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1025..1073
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1098..1117
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(1157..1198)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    1224..1248
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1624..1648)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1677..1696)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1678..1696)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1741..1944)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1770..1790)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1945..2324
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2496..2515
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2590..2694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2695..2763,2764..3555)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPKLNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2913..2932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3726..4314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4215..4234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4468..4485
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4602..4623
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4638..4643,4644..4661,4662..4668)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4676..4692
                     /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     4681..4703
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4700..4716
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4700..4716
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4765..4886
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4776..4795)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4830..4849
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5044..5439)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(join(5440..6150,6151..6153,6154..6156))
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5482..5503)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     5831..5850
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6090..6111
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(6175..6222)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(join(6236..6355,1..380))
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6284..6303)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
ORIGIN
        1 gagcccgccc ccggacccac cccttcccag cctctgagcc cagaaagcga aggagcaaag
       61 ctgctattgg ccgctgcccc aaaggcctac ccgcttccat tgctcagcgg tgctgtccat
      121 ctgcacgaga ctagtgagac gtgctacttc catttgtcac gtcctgcacg acgcgagctg
      181 cggggcgggg gggaacttcc tgactagggg aggagtagaa ggtggcgcga aggggccacc
      241 aaagaacgga gccggttggc gcctaccggt ggatgtggaa tgtgtgcgag gccagaggcc
      301 acttgtgtag cgccaagtgc ccagcggggc tgctaaagcg catgctccag actgccttgg
      361 gaaaagcgcc tcccctaccc ggtagctcga ggataattcg cgttaaattt ttgttaaatc
      421 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
      481 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
      541 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
      601 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
      661 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
      721 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
      781 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtggggata ccccctagag
      841 ccccagctgc gcagatctgc tatggcaggg cctgccgccc cgacgttggc tgcgagccct
      901 gggccttcac ccgaacttgg ggggtggggt ggggaaaagg aagaaacgcg ggcgtattgg
      961 ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc cctgggaccg aaccccgcgt
     1021 ttatgaacaa acgacccaac acccgtgcgt tttattctgt ctttttattg ccgtcatagc
     1081 gcgggttcct tccggtattg tctccttccg tgtttcagtt agcctccccc gtttaaactc
     1141 attactaacc ggtacgcgta gaatcgagac cgaggagagg gttagggata ggcttacctt
     1201 cgaaccgcgg gccctctaga tcaaccactt tgtacaagaa agctgggttt cagggggaca
     1261 ttttgaaccc agccgcacca atgttctcat ggatggtggc cttcgattct tctagattca
     1321 agatttggct cttgtacttt tcctgccagt cctctacaat gtactggtgg taggggtcat
     1381 tggagtgttc ccgtctcttg gatttcacag tgctcaccag gatggccacg atgatgaaag
     1441 agaacattcc aatcatcacc atgaggtaca ggatgacata gtagaagttc tcagcatcaa
     1501 ctttggcttg gagggcctct tgctcagctg ttgtgttctg gcgccaattg tccatataag
     1561 taataaaaat ccttcggaag acgtcttcca gcgtctgtgt gaaattggat aaagtagaca
     1621 tggtgcctgc ttttttgtac aaacttgttg atagcttaac tagccagctt gggtctccct
     1681 atagtgagtc gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag
     1741 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc
     1801 ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt
     1861 gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt
     1921 gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg
     1981 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc
     2041 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     2101 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     2161 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
     2221 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
     2281 ccccgtaatt gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc
     2341 ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaattgtc
     2401 ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa
     2461 cacacaagca gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     2521 agtgcaccat aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg
     2581 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2641 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2701 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2761 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2821 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2881 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     2941 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3001 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3061 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3121 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3181 tgggaaccga agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     3241 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3301 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3361 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     3421 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     3481 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     3541 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3601 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3661 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3721 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3781 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3841 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3901 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3961 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4021 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4081 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4141 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4201 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4261 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4321 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4381 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     4441 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     4501 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     4561 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     4621 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     4681 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta
     4741 gaggtcgacg gtatacagac atgataagat acattgatga gtttggacaa accacaacta
     4801 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     4861 ccattataag ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg
     4921 cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag
     4981 aaggcggcgg tggaatcgaa atctcgtagc acgtgtcagt cctgctcctc ggccacgaag
     5041 tgcttagccc tcccacacat aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg
     5101 ctgtccatca ctgtccttca ctatggcttt gatcccagga tgcagatcga gaagcacctg
     5161 tcggcaccgt ccgcaggggc tcaagatgcc cctgttctca tttccgatcg cgacgataca
     5221 agtcaggttg ccagctgccg cagcagcagc agtgcccagc accacgagtt ctgcacaagg
     5281 tcccccagta aaatgatata cattgacacc agtgaagatg cggccgtcgc tagagagagc
     5341 tgcgctggcg acgctgtagt cttcagagat ggggatgctg ttgattgtag ccgttgctct
     5401 ttcaatgagg gtggattctt cttgagacaa aggcttggcc ttgtacagct cgtccatgcc
     5461 gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     5521 ggggtctttg ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg
     5581 gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc
     5641 gatgttgtgg cgggtcttga agttcacctt gatgccgttc ttctgcttgt cggcggtgat
     5701 atagaccttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc
     5761 cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac
     5821 ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta
     5881 gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcgggc
     5941 gaagcactgc acgccgtagg tgaaggtggt cacgagggtg ggccagggca cgggcagctt
     6001 gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc
     6061 gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac
     6121 caccccggtg aacagctcct cgcccttgct caccatggtt tagttcctca ccttgtcgta
     6181 ttatactatg ccgatatact atgccgatga ttaattgtca acacgtgtct agagtcgaaa
     6241 ggcccggaga tgaggaagag gagaacagcg cggcagacgt gcgcttttga agcgtgcaga
     6301 atgccgggcc tccggaggac cttcgggcgc ccgccccgcc cctgagcccg cccct
//
