LOCUS       sequence-349770-        7222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding WDR4 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: WDR4_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    677..701
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1944..1968)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1994..2035
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2075..2094)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2119..2167
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2369..2797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2456..2475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2666..2687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2812..3311
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2847..2870)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3244..3263
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3325..3372
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3391..3393,3394..3396,3397..4107)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3436..3457)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3697..3716)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4044..4065
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4108..4503
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4661..4782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4698..4717)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4752..4771
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4831..4847)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4831..4847)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4844..4866)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4855..4871
                     /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(4879..4885,4886..4903,4904..4909))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4924..4945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5062..5079)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5233..5821)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5313..5332)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5992..6783,6784..6852))
                     /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"
     promoter        complement(6853..6957)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7032..7051)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc accatggcgg gctctgtggg
      721 actggcgttg tgcgggcaga cgttggtggt gcggggcggc agccgattcc tggccacctc
      781 catagcaagc agtgatgatg acagcctctt catctatgac tgcagtgctg cagaaaagaa
      841 gtcacaagaa aataaagggg aggacgcgcc cttggaccag gggagcggtg cgattctggc
      901 gtccaccttc tccaagtctg gcagctattt tgctttaacc gatgacagta agcgtctgat
      961 tcttttccgt acaaaaccat ggcaatgtct gagtgtcagg accgtggcaa ggaggtgtac
     1021 agccctgact ttcatagcct cggaggagaa ggtcttggtg gccgacaagt ctggagacgt
     1081 ctactccttt tcggtgctgg agccacacgg gtgtggccgt ctagagctgg gacacctgtc
     1141 tatgctgtta gatgtggctg tgagtcctga tgaccgcttc atcctcactg ccgaccggga
     1201 cgagaagatc cgagtcagct gggccgcggc gccccatagc atcgagtcct tctgcttggg
     1261 gcacacagag tttgtgagcc gtatctccgt ggtgccaact cagcccgggc tgcttctgtc
     1321 ctcctctggg gacggcaccc tgaggctctg ggagtacagg agcggccgcc agctgcactg
     1381 ctgtcacctg gccagtctgc aggagctggt ggacccccag gccccccaga agtttgccgc
     1441 gtccaggatt gcattctggt gccaggagaa ctgcgtggcg ctcctgtgcg acggcactcc
     1501 tgtggtctac atcttccagc tggacgcccg cagacagcag ttggtgtaca ggcagcagct
     1561 ggcgttccag caccaagtgt gggacgtggc tttcgaggag acccaggggc tgtgggtgct
     1621 ccaggactgc caggaagccc ccctggtgct ctacaggcct gtgggcgacc agtggcagtc
     1681 tgttcctgag agcaccgtgt taaagaaagt ctctggtgtt cttcgtggga actgggccat
     1741 gctggaaggc tctgccggcg cagacgccag cttcagcagt ctctacaagg ccacgttcga
     1801 caacgtgacc tcctacctga agaagaaaga ggagagactg cagcagcagc tagagaagaa
     1861 gcagcggcgc cggagtcccc cgcctgggcc cgacgggcat gccaagaaga tgagaccggg
     1921 ggaggcgacg ctaagttgct gaaacccagc tttcttgtac aaagtggttg atctagaggg
     1981 cccgcggttc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac
     2041 cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg
     2101 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg
     2161 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     2221 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     2281 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcagatctg
     2341 cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2401 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2461 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2521 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2581 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2641 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2701 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2761 aaaaatgagc tgatttaaca aaaatttaac gcgaattatc ctcgagctac cgggtagggg
     2821 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     2881 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     2941 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     3001 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     3061 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     3121 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     3181 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     3241 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     3301 cgggcctttc gactctagac acgtgttgac aattaatcat cggcatagta tatcggcata
     3361 gtataatacg acaaggtgag gaactaaacc atggtgagca agggcgagga gctgttcacc
     3421 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     3481 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3541 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcaccta cggcgtgcag
     3601 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3661 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3721 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3781 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaag
     3841 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg tgaacttcaa gacccgccac
     3901 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3961 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     4021 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     4081 actctcggca tggacgagct gtacaaggcc aagcctttgt ctcaagaaga atccaccctc
     4141 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     4201 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     4261 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     4321 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     4381 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat
     4441 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc
     4501 taagcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
     4561 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4621 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     4681 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     4741 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     4801 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4861 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4921 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4981 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5041 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5101 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5161 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5221 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5281 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5341 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5401 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5461 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5521 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5581 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5641 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     5701 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5761 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5821 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     5881 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5941 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6001 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6061 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6121 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6181 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6241 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6301 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagcttcgg
     6361 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6421 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6481 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6541 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6601 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     6661 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     6721 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     6781 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     6841 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6901 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6961 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7021 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     7081 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     7141 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     7201 gtacgggcca gatatacgcg tt
//
