LOCUS       sequence-349427-        7585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding PDE9A and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7585)
  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 7585)
  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: PDE9A_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    297..321
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1927..1951)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1977..2018
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2058..2077)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2102..2150
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2352..2780
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2439..2458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2649..2670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2795..3294
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2830..2853)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3227..3246
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3308..3355
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3374..3376,3377..3379,3380..4090)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3419..3440)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3680..3699)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4027..4048
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4091..4486
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4644..4765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4681..4700)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4735..4754
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4814..4830)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4814..4830)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4827..4849)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4838..4854
                     /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(4862..4868,4869..4886,4887..4892))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4907..4928
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5045..5062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5216..5804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5296..5315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5975..6766,6767..6835))
                     /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     6598..6617
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6836..6940)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7015..7034)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7206..7585
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagttaag ctatcaacaa
      301 gtttgtacaa aaaagcaggc accatgggat ccggctcctc cagctaccgg cccaaggcca
      361 tctacctgga catcgatgga cgcattcaga aggtaatctt cagcaagtac tgcaactcca
      421 gcgacatcat ggacctgttc tgcatcgcca ccggcctgcc tcggaacacg accatctccc
      481 tgctgaccac cgacgacgcc atggtctcca tcgaccccac catgcccgcg aattcagaac
      541 gcactccgta caaagtgaga cctgtggcca tcaagcaact ctccgagaga gaagaattaa
      601 tccagagcgt gctggcgcag gttgcagagc agttctcaag agcattcaaa atcaatgaac
      661 tgaaagctga agttgcaaat cacttggctg tcctagagaa acgcgtggaa ttggaaggac
      721 taaaagtggt ggagattgag aaatgcaaga gtgacattaa gaagatgagg gaggagctgg
      781 cggccagaag cagcaggacc aactgcccct gtaagtacag ttttttggat aaccacaaga
      841 agttgactcc tcgacgcgat gttcccactt accccaagta cctgctctct ccagagacca
      901 tcgaggccct gcggaagccg acctttgacg tctggctttg ggagcccaat gagatgctga
      961 gctgcctgga gcacatgtac cacgacctcg ggctggtcag ggacttcagc atcaaccctg
     1021 tcaccctcag gaggtggctg ttctgcgtcc acgacaacta cagaaacaac cccttccaca
     1081 acttccggca ctgcttctgc gtggcccaga tgatgtacag catggtctgg ctctgcagtc
     1141 tccaggagaa gttctcacaa acggatatcc tgatcctaat gacagcggcc atctgccacg
     1201 atctggacca tcccggctac aacaacacgt accagatcaa tgcccgcaca gagctggcgg
     1261 tccgctacaa tgacatctca ccgctggaga accaccactg cgccgtggcc ttccagatcc
     1321 tcgccgagcc tgagtgcaac atcttctcca acatcccacc tgatgggttc aagcagatcc
     1381 gacagggaat gatcacatta atcttggcca ctgacatggc aagacatgca gaaattatgg
     1441 attctttcaa agagaaaatg gagaattttg actacagcaa cgaggagcac atgaccctgc
     1501 tgaagatgat tttgataaaa tgctgtgata tctctaacga ggtccgtcca atggaagtcg
     1561 cagagccttg ggtggactgt ttattagagg aatattttat gcagagcgac cgtgagaagt
     1621 cagaaggcct tcccgtggcc ccgttcatgg accgagacaa agtgaccaag gccacagccc
     1681 agattgggtt catcaagttt gtcctgatcc caatgtttga aacagtgacc aagctcttcc
     1741 ccatggttga ggagatcatg ctgcagccac tttgggaatc ccgagatcgc tacgaggagc
     1801 tgaagcggat agatgacgcc atgaaagagt tacagaagaa gactgacagc ttgacgtctg
     1861 gggccaccga gaagtccaga gagagaagca gagatgtgaa aaacagtgaa ggagactgtg
     1921 cctagaaccc agctttcttg tacaaagtgg ttgatctaga gggcccgcgg ttcgaaggta
     1981 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggttag taatgagttt
     2041 aaacggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     2101 acggcaataa aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg
     2161 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     2221 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     2281 cgcagccaac gtcggggcgg caggccctgc catagcagat ctgcgcagct ggggctctag
     2341 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     2401 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     2461 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     2521 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     2581 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     2641 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2701 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2761 acaaaaattt aacgcgaatt atcctcgagc taccgggtag gggaggcgct tttcccaagg
     2821 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     2881 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     2941 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     3001 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     3061 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     3121 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     3181 gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt
     3241 caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgactcta
     3301 gacacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     3361 gaggaactaa accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     3421 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     3481 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3541 gccctggccc accctcgtga ccaccttcac ctacggcgtg cagtgcttcg cccgctaccc
     3601 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3661 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3721 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3781 catcctgggg cacaagctgg agtacaacta caacagccac aaggtctata tcaccgccga
     3841 caagcagaag aacggcatca aggtgaactt caagacccgc cacaacatcg aggacggcag
     3901 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3961 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     4021 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     4081 gctgtacaag gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     4141 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     4201 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     4261 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     4321 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     4381 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     4441 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagcac ttcgtggccg
     4501 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     4561 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4621 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4681 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4741 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4801 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4861 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4921 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4981 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5041 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5101 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5161 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5221 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5281 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5341 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5401 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5461 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5521 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5581 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5641 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5701 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5761 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5821 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5881 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5941 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6001 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6061 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6121 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6181 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6241 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6301 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctt cggttcccaa cgatcaaggc
     6361 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6421 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6481 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6541 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6601 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6661 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6721 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6781 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6841 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6901 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6961 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7021 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7081 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     7141 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7201 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     7261 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     7321 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     7381 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     7441 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     7501 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     7561 cgtattagtc atcgctatta ccatg
//
