LOCUS       sequence-350512-        7222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding GNAS and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7222)
  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 7222)
  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: GNAS_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7222
                     /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(2178..2202)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2228..2269
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2309..2328)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2353..2401
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2603..3031
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2690..2709)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2900..2921
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3046..3545
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3081..3104)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3478..3497
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3559..3606
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3625..3627,3628..3630,3631..4341)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3670..3691)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3931..3950)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4278..4299
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4342..4737
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4895..5016
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4932..4951)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4986..5005
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5065..5081)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5065..5081)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5078..5100)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5089..5105
                     /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(5113..5119,5120..5137,5138..5143))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5158..5179
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5296..5313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5467..6055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5547..5566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6226..7017,7018..7086))
                     /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     6849..6868
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7087..7191)
                     /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 aggctccgcg gccgccccct tcaccatggg
      961 ctgcctcggg aacagtaaga ccgaggacca gcgcaacgag gagaaggcgc agcgtgaggc
     1021 caacaaaaag atcgagaagc agctgcagaa ggacaagcag gtctaccggg ccacgcaccg
     1081 cctgctgctg ctgggtgctg gagaatctgg taaaagcacc attgtgaagc agatgaggat
     1141 cctgcatgtt aatgggttta atggagaggg cggcgaagag gacccgcagg ctgcaaggag
     1201 caacagcgat ggcagtgaga aggcaaccaa agtgcaggac atcaaaaaca acctgaaaga
     1261 ggcgattgaa accattgtgg ccgccatgag caacctggtg ccccccgtgg agctggccaa
     1321 ccccgagaac cagttcagag tggactacat cctgagtgtg atgaacgtgc ctgactttga
     1381 cttccctccc gaattctatg agcatgccaa ggctctgtgg gaggatgaag gagtgcgtgc
     1441 ctgctacgaa cgctccaacg agtaccagct gattgactgt gcccagtact tcctggacaa
     1501 gatcgacgtg atcaagcagg ctgactatgt gccgagcgat caggacctgc ttcgctgccg
     1561 tgtcctgact tctggaatct ttgagaccaa gttccaggtg gacaaagtca acttccacat
     1621 gtttgacgtg ggtggccagc gcgatgaacg ccgcaagtgg atccagtgct tcaacgatgt
     1681 gactgccatc atcttcgtgg tggccagcag cagctacaac atggtcatcc gggaggacaa
     1741 ccagaccaac cgcctgcagg aggctctgaa cctcttcaag agcatctgga acaacagatg
     1801 gctgcgcacc atctctgtga tcctgttcct caacaagcaa gatctgctcg ctgagaaagt
     1861 ccttgctggg aaatcgaaga ttgaggacta ctttccagaa tttgctcgct acactactcc
     1921 tgaggatgct actcccgagc ccggagagga cccacgcgtg acccgggcca agtacttcat
     1981 tcgagatgag tttctgagga tcagcactgc cagtggagat gggcgtcact actgctaccc
     2041 tcatttcacc tgcgctgtgg acactgagaa catccgccgt gtgttcaacg actgccgtga
     2101 catcattcag cgcatgcacc ttcgtcagta cgagctgctc tactagccaa ctttcttgta
     2161 caagggtggg cgcgccgacc cagctttctt gtacaaagtg gttgatctag agggcccgcg
     2221 gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc gtaccggtta
     2281 gtaatgagtt taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     2341 cccgcgctat gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt
     2401 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     2461 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     2521 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgcagc
     2581 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2641 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2701 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2761 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2821 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2881 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2941 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3001 gagctgattt aacaaaaatt taacgcgaat tatcctcgag ctaccgggta ggggaggcgc
     3061 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     3121 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     3181 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     3241 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     3301 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     3361 agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg
     3421 ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat
     3481 tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc
     3541 tttcgactct agacacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3601 tacgacaagg tgaggaacta aaccatggtg agcaagggcg aggagctgtt caccggggtg
     3661 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     3721 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3781 aagctgcccg tgccctggcc caccctcgtg accaccttca cctacggcgt gcagtgcttc
     3841 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3901 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3961 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4021 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caaggtctat
     4081 atcaccgccg acaagcagaa gaacggcatc aaggtgaact tcaagacccg ccacaacatc
     4141 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     4201 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     4261 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     4321 ggcatggacg agctgtacaa ggccaagcct ttgtctcaag aagaatccac cctcattgaa
     4381 agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca
     4441 gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga
     4501 ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact
     4561 tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga
     4621 caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag
     4681 ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaagca
     4741 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
     4801 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4861 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4921 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4981 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     5041 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     5101 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     5161 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     5221 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5281 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5341 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5401 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5461 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5521 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5581 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5641 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5701 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5761 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5821 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5881 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5941 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6001 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6061 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6121 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6181 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6241 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6301 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6361 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6421 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6481 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6541 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct tcggttccca
     6601 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6661 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6721 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6781 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6841 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6901 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6961 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7021 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7081 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7141 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7201 ccgaaaagtg ccacctgacg tc
//
