LOCUS       sequence-349720-        6805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding CBR3 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6805)
  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 6805)
  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: CBR3_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6805
                     /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(1539..1563)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1589..1630
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1670..1689)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1714..1762
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1964..2392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2051..2070)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2261..2282
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2407..2894
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2442..2465)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2827..2846
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2908..2955
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2974..2976,2977..2979,2980..3690)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3019..3040)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3280..3299)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3627..3648
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3691..4086
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4244..4365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4281..4300)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4335..4354
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4414..4430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4414..4430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4427..4449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4438..4454
                     /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(4462..4468,4469..4486,4487..4492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4507..4528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4645..4662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4816..5404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4896..4915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5575..6366,6367..6435))
                     /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     6198..6217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6436..6540)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6615..6634)
                     /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 accatgtcgt cctgcagccg
      721 cgtggcgctg gtgaccgggg ccaacagggg catcggcttg gccatcgcgc gcgaactgtg
      781 ccgacagttc tctggggatg tggtgctcac cgcgcgggac gtggcgcggg gccaggcggc
      841 cgtgcagcag ctgcaggcgg agggcctgag cccgcgcttc caccaactgg acatcgacga
      901 cttgcagagc atccgcgccc tgcgcgactt cctgcgcaag gagtacgggg ggctcaatgt
      961 actggtcaac aacgcggccg tcgccttcaa gagtgatgat ccaatgccct ttgacattaa
     1021 agctgagatg acactgaaga caaatttttt tgccactaga aacatgtgca acgagttact
     1081 gccgataatg aaacctcatg ggagagtggt gaatatcagt agtttgcagt gtttaagggc
     1141 ttttgaaaac tgcagtgaag atctgcagga aaggttccac agtgagacac tcacagaagg
     1201 agacctggtg gatctcatga aaaagtttgt ggaggacaca aaaaatgagg tgcatgagag
     1261 ggaaggctgg cccaactcac cttatggggt gtccaagttg ggggtcacag tcttatcgag
     1321 gatcctggcc aggcgtctgg atgagaagag gaaagctgac aggattctgg tgaatgcgtg
     1381 ctgcccagga ccagtgaaga cagacatgga tgggaaagac agcatcagga ctgtggagga
     1441 gggggctgag acccctgtct acttggccct cttgcctcca gatgccactg agccacaagg
     1501 ccagttggtc catgacaaag ttgtgcaaaa ctggtagaac ccagctttct tgtacaaagt
     1561 ggttgatcta gagggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct
     1621 cgattctacg cgtaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga
     1681 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     1741 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     1801 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     1861 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     1921 gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     1981 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2041 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2101 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2161 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2221 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2281 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2341 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttatcctcga
     2401 gctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     2461 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     2521 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     2581 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     2641 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     2701 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     2761 gaaggggtgg gtccgggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
     2821 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     2881 ctccgggcct ttcgactcta gacacgtgtt gacaattaat catcggcata gtatatcggc
     2941 atagtataat acgacaaggt gaggaactaa accatggtga gcaagggcga ggagctgttc
     3001 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3061 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3121 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac ctacggcgtg
     3181 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3241 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3301 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3361 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3421 aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc
     3481 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3541 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3601 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3661 atcactctcg gcatggacga gctgtacaag gccaagcctt tgtctcaaga agaatccacc
     3721 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     3781 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     3841 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     3901 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     3961 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     4021 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
     4081 ggctaagcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
     4141 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4201 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4261 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4321 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4381 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4441 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4501 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4561 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4621 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4681 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4741 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4801 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4861 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4921 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4981 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5041 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5101 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5161 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5221 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5281 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5341 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5401 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5461 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5521 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5581 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5641 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5701 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5761 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5821 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5881 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctt
     5941 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6001 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6061 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6121 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6181 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6241 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6301 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6361 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6421 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6481 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6541 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6601 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6661 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6721 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     6781 gatgtacggg ccagatatac gcgtt
//
