LOCUS       sequence-349707-        8245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding APP and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8245)
  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 8245)
  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: APP_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..8245
                     /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"
     CDS             complement(654..665)
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="NHKV"
     CDS             1145..1165
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     protein_bind    complement(2587..2611)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2637..2678
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2718..2737)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2762..2810
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3012..3440
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3099..3118)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3309..3330
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3455..3954
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3490..3513)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3887..3906
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3968..4015
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(4034..4036,4037..4039,4040..4750)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4079..4100)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4340..4359)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4687..4708
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4751..5146
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5304..5425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5341..5360)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5395..5414
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5474..5490)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5474..5490)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5487..5509)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5498..5514
                     /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(5522..5528,5529..5546,5547..5552))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5567..5588
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5705..5722)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5876..6464)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5956..5975)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6635..7426,7427..7495))
                     /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     7258..7277
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7496..7600)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7675..7694)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7866..8245
                     /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 tccaccatgc tgcccggttt ggcactgctc ctgctggccg
      361 cctggacggc tcgggcgctg gaggtaccca ctgatggtaa tgctggcctg ctggctgaac
      421 cccagattgc catgttctgt ggcagactga acatgcacat gaatgtccag aatgggaagt
      481 gggattcaga tccatcaggg accaaaacct gcattgatac caaggaaggc atcctgcagt
      541 attgccaaga agtctaccct gaactgcaga tcaccaatgt ggtagaagcc aaccaaccag
      601 tgaccatcca gaactggtgc aagcggggcc gcaagcagtg caagacccat ccccactttg
      661 tgattcccta ccgctgctta gttggtgagt ttgtaagtga tgcccttctc gttcctgaca
      721 agtgcaaatt cttacaccag gagaggatgg atgtttgcga aactcatctt cactggcaca
      781 ccgtcgccaa agagacatgc agtgagaaga gtaccaactt gcatgactac ggcatgttgc
      841 tgccctgcgg aattgacaag ttccgagggg tagagtttgt gtgttgccca ctggctgaag
      901 aaagtgacaa tgtggattct gctgatgcgg aggaggatga ctcggatgtc tggtggggcg
      961 gagcagacac agactatgca gatgggagtg aagacaaagt agtagaagta gcagaggagg
     1021 aagaagtggc tgaggtggaa gaagaagaag ccgatgatga cgaggacgat gaggatggtg
     1081 atgaggtaga ggaagaggct gaggaaccct acgaagaagc cacagagaga accaccagca
     1141 ttgccaccac caccaccacc accacagagt ctgtggaaga ggtggttcga gaggtgtgct
     1201 ctgaacaagc cgagacgggg ccgtgccgag caatgatctc ccgctggtac tttgatgtga
     1261 ctgaagggaa gtgtgcccca ttcttttacg gcggatgtgg cggcaaccgg aacaactttg
     1321 acacagaaga gtactgcatg gccgtgtgtg gcagcgccat tcctacaaca gcagccagta
     1381 cccctgatgc cgttgacaag tatctcgaga cacctgggga tgagaatgaa catgcccatt
     1441 tccagaaagc caaagagagg cttgaggcca agcaccgaga gagaatgtcc caggtcatga
     1501 gagaatggga agaggcagaa cgtcaagcaa agaacttgcc taaagctgat aagaaggcag
     1561 ttatccagca tttccaggag aaagtggaat ctttggaaca ggaagcagcc aacgagagac
     1621 agcagctggt ggagacacac atggccagag tggaagccat gctcaatgac cgccgccgcc
     1681 tggccctgga gaactacatc accgctctgc aggctgttcc tcctcggcct cgtcacgtgt
     1741 tcaatatgct aaagaagtat gtccgcgcag aacagaagga cagacagcac accctaaagc
     1801 atttcgagca tgtgcgcatg gtggatccca agaaagccgc tcagatccgg tcccaggtta
     1861 tgacacacct ccgtgtgatt tatgagcgca tgaatcagtc tctctccctg ctctacaacg
     1921 tgcctgcagt ggccgaggag attcaggatg aagttgatga gctgcttcag aaagagcaaa
     1981 actattcaga tgacgtcttg gccaacatga ttagtgaacc aaggatcagt tacggaaacg
     2041 atgctctcat gccatctttg accgaaacga aaaccaccgt ggagctcctt cccgtgaatg
     2101 gagagttcag cctggacgat ctccagccgt ggcattcttt tggggctgac tctgtgccag
     2161 ccaacacaga aaacgaagtt gagcctgttg atgcccgccc tgctgccgac cgaggactga
     2221 ccactcgacc aggttctggg ttgacaaata tcaagacgga ggagatctct gaagtgaaga
     2281 tggatgcaga attccgacat gactcaggat atgaagttca tcatcaaaaa ttggtgttct
     2341 ttgcagaaga tgtgggttca aacaaaggtg caatcattgg actcatggtg ggcggtgttg
     2401 tcatagcgac agtgatcgtc atcaccttgg tgatgctgaa gaagaaacag tacacatcca
     2461 ttcatcatgg tgtggtggag gttgacgccg ctgtcacccc agaggagcgc cacctgtcca
     2521 agatgcagca gaacggctac gaaaatccaa cctacaagtt ctttgagcag atgcagaact
     2581 gaatccaccc agctttcttg tacaaagtgg ttgatctaga gggcccgcgg ttcgaaggta
     2641 agcctatccc taaccctctc ctcggtctcg attctacgcg taccggttag taatgagttt
     2701 aaacggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     2761 acggcaataa aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg
     2821 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     2881 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     2941 cgcagccaac gtcggggcgg caggccctgc catagcagat ctgcgcagct ggggctctag
     3001 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3061 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3421 acaaaaattt aacgcgaatt atcctcgagc taccgggtag gggaggcgct tttcccaagg
     3481 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     3541 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     3601 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     3661 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     3721 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     3781 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     3841 gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt
     3901 caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgactcta
     3961 gacacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     4021 gaggaactaa accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     4081 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     4141 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     4201 gccctggccc accctcgtga ccaccttcac ctacggcgtg cagtgcttcg cccgctaccc
     4261 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     4321 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     4381 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     4441 catcctgggg cacaagctgg agtacaacta caacagccac aaggtctata tcaccgccga
     4501 caagcagaag aacggcatca aggtgaactt caagacccgc cacaacatcg aggacggcag
     4561 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     4621 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     4681 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     4741 gctgtacaag gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     4801 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     4861 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     4921 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     4981 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     5041 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     5101 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagcac ttcgtggccg
     5161 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     5221 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5281 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     5341 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5401 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     5461 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5521 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5581 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5641 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5701 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5761 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5821 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5881 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5941 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6001 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6061 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6121 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6181 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6241 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6301 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6361 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6421 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6481 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6541 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6601 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6661 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6721 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6781 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6841 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6901 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6961 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctt cggttcccaa cgatcaaggc
     7021 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7081 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7141 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7201 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7261 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7321 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7381 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7441 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7501 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7561 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7621 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     7681 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     7741 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     7801 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     7861 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     7921 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     7981 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     8041 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     8101 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     8161 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     8221 cgtattagtc atcgctatta ccatg
//
