LOCUS       sequence-349428-        6723 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding DsRed2 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6723)
  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 6723)
  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: DsRed2_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        90..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     718..737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        718..736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    766..790
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      828..837
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             834..1511
                     /codon_start=1
                     /product="improved tetrameric variant of DsRed fluorescent 
                     protein"
                     /label=DsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(1014..1034)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1426..1449
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(1534..1558)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1584..1625
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1665..1684)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1709..1757
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1959..2387
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2046..2065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2256..2277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2402..2901
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2437..2460)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2834..2853
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2915..2962
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2981..2983,2984..2986,2987..3697)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3026..3047)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3287..3306)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3634..3655
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3698..4093
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4251..4372
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4288..4307)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4342..4361
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4421..4437)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4421..4437)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4434..4456)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4445..4461
                     /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(4469..4475,4476..4493,4494..4499))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4514..4535
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4652..4669)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4823..5411)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4903..4922)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5582..6373,6374..6442))
                     /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     6205..6224
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6443..6547)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6622..6641)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
       61 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
      721 tacgactcac tatagggaga cccaagctgg ctagttaagc tatcaacaag tttgtacaaa
      781 aaagcaggct ttaaaggaac caattcagtc gactggatcc accggtcgcc accatggcct
      841 cctccgagaa cgtcatcacc gagttcatgc gcttcaaggt gcgcatggag ggcaccgtga
      901 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggccacaaca
      961 ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc
     1021 cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca
     1081 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     1141 gcgtggcgac cgtgacccag gactcctccc tgcaggacgg ctgcttcatc tacaaggtga
     1201 agttcatcgg cgtgaacttc ccctccgacg gccccgtgat gcagaagaag accatgggct
     1261 gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagacccaca
     1321 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg
     1381 ccaagaagcc cgtgcagctg cccggctact actacgtgga cgccaagctg gacatcacct
     1441 cccacaacga ggactacacc atcgtggagc agtacgagcg caccgagggc cgccaccacc
     1501 tgttcctgta gcggccgcac tcgagatatc tagacccagc tttcttgtac aaagtggttg
     1561 atctagaggg cccgcggttc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt
     1621 ctacgcgtac cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag
     1681 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg
     1741 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     1801 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     1861 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     1921 agcagatctg cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     1981 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2041 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2101 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2161 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2221 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2281 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2341 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattatc ctcgagctac
     2401 cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg
     2461 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg
     2521 cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc
     2581 aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca
     2641 cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt
     2701 tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg
     2761 ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc
     2821 tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct
     2881 tcctcatctc cgggcctttc gactctagac acgtgttgac aattaatcat cggcatagta
     2941 tatcggcata gtataatacg acaaggtgag gaactaaacc atggtgagca agggcgagga
     3001 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3061 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3121 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcaccta
     3181 cggcgtgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     3241 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3301 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3361 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3421 cagccacaag gtctatatca ccgccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3481 gacccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3541 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     3601 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3661 cgccgggatc actctcggca tggacgagct gtacaaggcc aagcctttgt ctcaagaaga
     3721 atccaccctc attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta
     3781 cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata
     3841 tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc
     3901 agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc
     3961 ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa
     4021 ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt
     4081 gtgggagggc taagcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
     4141 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4201 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4261 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4321 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4381 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4441 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4501 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4561 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4621 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4681 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4741 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4801 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4861 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4921 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4981 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5041 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5101 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5161 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5221 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5281 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5341 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5401 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5461 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5521 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5581 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5641 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5701 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5761 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5821 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5881 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5941 tcagcttcgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6001 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6061 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6121 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6181 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6241 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6301 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6361 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6421 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6481 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6541 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     6601 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     6661 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     6721 aag
//
