LOCUS       sequence-350534-        7111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding KCNJ15 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7111)
  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 7111)
  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: KCNJ15_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7111
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2067..2091)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2117..2158
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2198..2217)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2242..2290
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2492..2920
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2579..2598)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2789..2810
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2935..3434
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2970..2993)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3367..3386
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3448..3495
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3514..3516,3517..3519,3520..4230)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3559..3580)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3820..3839)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4167..4188
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4231..4626
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4784..4905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4821..4840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4875..4894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4954..4970)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4954..4970)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4967..4989)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4978..4994
                     /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)."
     promoter        complement(join(5002..5008,5009..5026,5027..5032))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5047..5068
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5185..5202)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5356..5944)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5436..5455)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6115..6906,6907..6975))
                     /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     6738..6757
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6976..7080)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggcaccatg gatgccattc acatcggcat
      961 gtccagcacc cccctggtga agcacactgc tggggctggg ctcaaggcca acagaccccg
     1021 cgtcatgtcc aagagtgggc acagcaacgt gagaattgac aaagtggatg gcatatacct
     1081 actctacctg caagacctgt ggaccacagt tatcgacatg aagtggagat acaaactcac
     1141 cctgttcgct gccacttttg tgatgacctg gttccttttt ggagtcatct actatgccat
     1201 cgcgtttatt catggggact tagaacccgg tgagcccatt tcaaatcata ccccctgcat
     1261 catgaaagtg gactctctca ctggggcgtt tctcttttcc ctggaatccc agacaaccat
     1321 tggctatgga gtccgttcca tcacagagga atgtcctcat gccatcttcc tgttggttgc
     1381 tcagttggtc atcacgacct tgattgagat cttcatcacc ggaaccttcc tggccaaaat
     1441 cgccagaccc aaaaagcggg ctgagaccat caagttcagc cactgtgcag tcatcaccaa
     1501 gcagaatggg aagctgtgct tggtgattca ggtagccaat atgaggaaga gcctcttgat
     1561 tcagtgccag ctctctggca agctcctgca gacccacgtc accaaggagg gggagcggat
     1621 tctcctcaac caagccactg tcaaattcca cgtggactcc tcctctgaga gccccttcct
     1681 cattctgccc atgacattct accatgtgct ggatgagacg agccccctga gagacctcac
     1741 accccaaaac ctaaaggaga aggagtttga gcttgtggtc ctcctcaatg ccactgtgga
     1801 atccaccagc gctgtctgcc agagccgaac atcttatatc ccagaggaaa tctactgggg
     1861 ttttgagttt gtgcctgtgg tatctctctc caaaaatgga aaatatgtgg ctgatttcag
     1921 tcagtttgaa cagattcgga aaagcccaga ttgcacattt tactgtgcag attctgagaa
     1981 acagcaactc gaggagaagt acaggcagga ggatcagagg gaaagagaac tgaggacact
     2041 tttattacaa cagagcaatg tctgaaaccc agctttcttg tacaaagtgg ttgatctaga
     2101 gggcccgcgg ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg
     2161 taccggttag taatgagttt aaacggggga ggctaactga aacacggaag gagacaatac
     2221 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg
     2281 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     2341 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     2401 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcagat
     2461 ctgcgcagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2521 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2581 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2641 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2701 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2761 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2821 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2881 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt atcctcgagc taccgggtag
     2941 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     3001 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     3061 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     3121 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     3181 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     3241 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     3301 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
     3361 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     3421 ctccgggcct ttcgactcta gacacgtgtt gacaattaat catcggcata gtatatcggc
     3481 atagtataat acgacaaggt gaggaactaa accatggtga gcaagggcga ggagctgttc
     3541 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3601 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3661 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac ctacggcgtg
     3721 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3781 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3841 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3901 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3961 aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc
     4021 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     4081 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     4141 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     4201 atcactctcg gcatggacga gctgtacaag gccaagcctt tgtctcaaga agaatccacc
     4261 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     4321 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     4381 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     4441 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     4501 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     4561 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
     4621 ggctaagcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
     4681 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4741 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4801 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4861 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4921 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4981 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5041 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5101 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5161 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5221 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5281 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5341 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5401 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5461 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5521 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5581 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5641 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5701 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5761 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5821 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5881 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5941 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6001 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6061 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6121 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6181 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6241 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6301 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6361 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6421 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctt
     6481 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6541 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6601 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6661 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6721 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6781 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6841 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6901 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6961 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7021 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7081 cacatttccc cgaaaagtgc cacctgacgt c
//
