LOCUS       sequence-349729-        7348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding GABPA and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  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 7348)
  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: GABPA_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7348
                     /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(1690..1714)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1740..1781
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1821..1840)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1865..1913
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2115..2543
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2202..2221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2412..2433
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2558..3057
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2593..2616)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2990..3009
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3071..3118
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3137..3139,3140..3142,3143..3853)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3182..3203)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3443..3462)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3790..3811
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3854..4249
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4407..4528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4444..4463)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4498..4517
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4577..4593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4577..4593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4590..4612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4601..4617
                     /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(4625..4631,4632..4649,4650..4655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4670..4691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4808..4825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4979..5567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5059..5078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5738..6529,6530..6598))
                     /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     6361..6380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6599..6703)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6778..6797)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6969..7348
                     /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 accatgacta aaagagaagc agaggagctg atagaaattg
      361 agattgatgg aacagagaaa gcagagtgca cagaagaaag cattgtagaa caaacctacg
      421 cgccagctga atgtgtaagc caggccatag acatcaatga accaataggc aatttaaaga
      481 aactgctaga accaagacta cagtgttctt tggatgctca tgaaatttgt ctgcaagata
      541 tccagctgga tccagaacga agtttatttg accaaggagt aaaaacagat ggaactgtac
      601 agcttagtgt acaggtaatt tcttaccaag gaattgaacc aaagttaaac atccttgaaa
      661 ttgttaaacc tgcggacact gttgaggttg ttattgatcc agatgcccac catgctgaat
      721 cagaagcaca tcttgttgaa gaagctcaag tgataactct tgatggcaca aaacacatca
      781 caaccatttc agatgaaact tcagaacaag tgacaagatg ggctgctgca ctggaaggct
      841 ataggaaaga acaagaacgc cttgggatac cctatgatcc catacagtgg tccacagacc
      901 aagtcctgca ttgggtggtt tgggtaatga aggaattcag catgaccgat atagacctca
      961 ccacactcaa catttcgggg agagaattat gtagtctcaa ccaagaagat ttttttcagc
     1021 gggttcctcg gggagaaatt ctctggagtc atctggaact tctccgaaaa tatgtattgg
     1081 caagtcaaga acaacagatg aatgaaatag ttacaattga tcaacctgtg caaattattc
     1141 cagcatcagt gcaatctgct acacctacta ccattaaagt tataaatagt agtgcgaaag
     1201 cagccaaagt acaaagagcg ccgaggattt caggagaaga tagaagctca cctgggaaca
     1261 gaacaggaaa caatggccaa atccaactat ggcagttttt gctagaactt cttactgata
     1321 aggacgctcg agactgcatt tcttgggttg gtgatgaagg tgaatttaag ctaaatcagc
     1381 ctgaactggt tgcacagaaa tggggacagc gtaaaaataa gcctacgatg aactatgaga
     1441 aactcagtcg tgcattaaga tattattacg atggggacat gatttgtaaa gttcaaggca
     1501 agagatttgt gtacaagttt gtctgtgact tgaagactct tattggatac agtgcagcgg
     1561 agttgaaccg tttggtcaca gaatgtgaac agaagaaact tgcaaagatg cagctccatg
     1621 gaattgccca gccagtcaca gcagtagctc tgtctactgc ttctctgcaa acggaaaagg
     1681 ataattagaa cccagctttc ttgtacaaag tggttgatct agagggcccg cggttcgaag
     1741 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt tagtaatgag
     1801 tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     1861 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg
     1921 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     1981 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     2041 gctcgcagcc aacgtcgggg cggcaggccc tgccatagca gatctgcgca gctggggctc
     2101 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2161 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     2221 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     2281 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     2341 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     2401 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2461 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2521 ttaacaaaaa tttaacgcga attatcctcg agctaccggg taggggaggc gcttttccca
     2581 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
     2641 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
     2701 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
     2761 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
     2821 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
     2881 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
     2941 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     3001 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgact
     3061 ctagacacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
     3121 ggtgaggaac taaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3181 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3241 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3301 cgtgccctgg cccaccctcg tgaccacctt cacctacggc gtgcagtgct tcgcccgcta
     3361 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3421 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3481 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3541 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaaggtct atatcaccgc
     3601 cgacaagcag aagaacggca tcaaggtgaa cttcaagacc cgccacaaca tcgaggacgg
     3661 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3721 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     3781 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3841 cgagctgtac aaggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     3901 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     3961 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     4021 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     4081 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     4141 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     4201 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg
     4261 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
     4321 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4381 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4441 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4501 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4561 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4621 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4681 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4741 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4801 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4861 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4921 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4981 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5041 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5101 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5161 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5221 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5281 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5341 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5401 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5461 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5521 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5581 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5641 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5701 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5761 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5821 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5881 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5941 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6001 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6061 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag cttcggttcc caacgatcaa
     6121 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6181 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6241 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6301 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6361 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6421 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6481 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6541 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6601 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6661 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6721 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     6781 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     6841 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     6901 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     6961 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     7021 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     7081 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     7141 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     7201 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     7261 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     7321 ctacgtatta gtcatcgcta ttaccatg
//
