LOCUS       sequence-349706-        6262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding S100B and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6262)
  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 6262)
  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: S100B_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6262
                     /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"
     regulatory      723..732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(984..1008)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1034..1075
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1115..1134)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1159..1207
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1409..1837
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1496..1515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1706..1727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1852..2351
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1887..1910)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2284..2303
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2365..2412
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2431..2433,2434..2436,2437..3147)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2476..2497)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2737..2756)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3084..3105
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3148..3543
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3701..3822
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3738..3757)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3792..3811
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3871..3887)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3871..3887)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3884..3906)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3895..3911
                     /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(3919..3925,3926..3943,3944..3949))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3964..3985
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4102..4119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4273..4861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4353..4372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5032..5823,5824..5892))
                     /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     5655..5674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5893..5997)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6072..6091)
                     /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 accatgtctg agctggagaa
      721 ggccatggtg gccctcatcg acgttttcca ccaatattct ggaagggagg gagacaagca
      781 caagctgaag aaatccgaac tcaaggagct catcaacaat gagctttccc atttcttaga
      841 ggaaatcaaa gagcaggagg ttgtggacaa agtcatggaa acactggaca atgatggaga
      901 cggcgaatgt gacttccagg aattcatggc ctttgttgcc atggttacta ctgcctgcca
      961 cgagttcttt gaacatgagt agaacccagc tttcttgtac aaagtggttg atctagaggg
     1021 cccgcggttc gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac
     1081 cggttagtaa tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg
     1141 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg
     1201 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     1261 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     1321 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcagatctg
     1381 cgcagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     1441 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1501 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1561 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1621 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1681 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1741 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1801 aaaaatgagc tgatttaaca aaaatttaac gcgaattatc ctcgagctac cgggtagggg
     1861 aggcgctttt cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc
     1921 gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg
     1981 ctccgttctt tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc
     2041 cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta
     2101 gtctcgtgca gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg
     2161 gccaatagca gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg
     2221 ggggcgggct caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc
     2281 cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc
     2341 cgggcctttc gactctagac acgtgttgac aattaatcat cggcatagta tatcggcata
     2401 gtataatacg acaaggtgag gaactaaacc atggtgagca agggcgagga gctgttcacc
     2461 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2521 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2581 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcaccta cggcgtgcag
     2641 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2701 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2761 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2821 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaag
     2881 gtctatatca ccgccgacaa gcagaagaac ggcatcaagg tgaacttcaa gacccgccac
     2941 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3001 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3061 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     3121 actctcggca tggacgagct gtacaaggcc aagcctttgt ctcaagaaga atccaccctc
     3181 attgaaagag caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc
     3241 agcgcagctc tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact
     3301 gggggacctt gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac
     3361 ctgacttgta tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg
     3421 tgccgacagg tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat
     3481 ggacagccga cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc
     3541 taagcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc
     3601 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3661 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3721 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3781 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3841 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3901 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3961 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4021 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4081 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4141 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4201 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4261 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4321 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4381 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4441 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4501 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4561 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4621 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4681 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4741 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4801 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4861 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4921 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4981 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5041 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5101 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5161 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5221 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5281 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5341 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagcttcgg
     5401 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5461 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5521 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5581 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5641 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5701 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5761 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5821 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5881 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5941 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6001 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     6061 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     6121 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     6181 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     6241 gtacgggcca gatatacgcg tt
//
