LOCUS       sequence-349713-        6943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding RWDD2B and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6943)
  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 6943)
  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: RWDD2B_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6943
                     /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(1285..1309)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1335..1376
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1416..1435)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1460..1508
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1710..2138
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1797..1816)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2007..2028
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2153..2652
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2188..2211)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2585..2604
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2666..2713
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2732..2734,2735..2737,2738..3448)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2777..2798)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3038..3057)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3385..3406
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3449..3844
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4002..4123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4039..4058)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4093..4112
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4172..4188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4172..4188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4185..4207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4196..4212
                     /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(4220..4226,4227..4244,4245..4250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4265..4286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4403..4420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4574..5162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4654..4673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5333..6124,6125..6193))
                     /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     5956..5975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6194..6298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6373..6392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6564..6943
                     /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 accatgaaaa ttgagctgtc catgcagcca tggaacccgg
      361 gttacagcag tgagggggcc acggctcaag aaacttacac atgtccaaaa atgattgaga
      421 tggagcaggc ggaggcccag cttgctgagt tagacctgct agccagtatg ttccctggtg
      481 agaatgagct catagtgaat gaccagctgg ctgtagcaga actgaaagat tgtattgaaa
      541 agaagacaat ggaggggcga tcttcaaaag tctactttac tatcaatatg aacctggatg
      601 tatctgacga aaaaatggcg atgttttctc tggcctgtat tcttcccttt aaatacccgg
      661 cagttctgcc tgaaatcact gtcagatcag tattattgag tagatcccag cagactcagc
      721 tgaacacaga tctgactgca ttcctgcaaa aacattgtca tggagatgtt tgtatactga
      781 atgccacaga gtgggttaga gaacacgcct ctggctatgt cagcagagat acttcatctt
      841 cacccaccac aggaagcaca gtccagtcag ttgacctcat cttcacgaga ctctggatct
      901 acagccatca tatctataac aaatgcaaaa gaaagaatat tctagagtgg gcaaaggagc
      961 tttccctgtc tgggtttagc atgcctggaa aacctggtgt tgtttgtgtg gaaggcccac
     1021 aaagtgcctg tgaagaattc tggtcaagac tcagaaaatt aaactggaag agaattttaa
     1081 ttcgccatcg agaagacatt ccttttgatg gtacaaatga tgaaacggaa agacaaagga
     1141 aattttccat ttttgaagaa aaagtgttca gtgttaatgg agccagggga aaccacatgg
     1201 actttggtca gctctatcag ttcttaaaca ccaaaggatg tggggatgtt ttccagatgt
     1261 tctttggtgt agaaggacaa tagaacccag ctttcttgta caaagtggtt gatctagagg
     1321 gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat tctacgcgta
     1381 ccggttagta atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg
     1441 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc
     1501 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     1561 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     1621 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct
     1681 gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1741 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1801 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1861 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1921 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1981 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2041 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2101 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattat cctcgagcta ccgggtaggg
     2161 gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg
     2221 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg
     2281 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
     2341 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
     2401 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
     2461 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc
     2521 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc
     2581 ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct
     2641 ccgggccttt cgactctaga cacgtgttga caattaatca tcggcatagt atatcggcat
     2701 agtataatac gacaaggtga ggaactaaac catggtgagc aagggcgagg agctgttcac
     2761 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2821 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2881 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca
     2941 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     3001 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     3061 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     3121 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     3181 ggtctatatc accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca
     3241 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     3301 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     3361 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     3421 cactctcggc atggacgagc tgtacaaggc caagcctttg tctcaagaag aatccaccct
     3481 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     3541 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     3601 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     3661 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     3721 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     3781 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     3841 ctaagcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     3901 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     3961 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
     4021 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4081 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
     4141 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4201 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4261 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4981 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagcttcg
     5701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6301 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     6361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     6421 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     6481 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     6541 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     6601 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     6661 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     6721 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     6781 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     6841 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     6901 tacttggcag tacatctacg tattagtcat cgctattacc atg
//
