LOCUS       sequence-349728-        7663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding CHAF1B and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7663)
  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 7663)
  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: CHAF1B_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7663
                     /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(2385..2409)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2435..2476
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2516..2535)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2560..2608
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2810..3238
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2897..2916)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3107..3128
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3253..3752
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3288..3311)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3685..3704
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3766..3813
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3832..3834,3835..3837,3838..4548)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3877..3898)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4138..4157)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4485..4506
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4549..4944
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5102..5223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5139..5158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5193..5212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5272..5288)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5272..5288)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5285..5307)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5296..5312
                     /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(5320..5326,5327..5344,5345..5350))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5365..5386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5503..5520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5674..6262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5754..5773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6433..7224,7225..7293))
                     /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     7056..7075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7294..7398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7473..7492)
                     /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 accatgaaag tcatcacttg
      721 tgaaatagcc tggcacaaca aggagcccgt gtacagcctg gacttccagc atgggacggc
      781 tgggaggatc cacagactgg cgtctgccgg cgtggacacc aatgtcagga tctggaaggt
      841 agaaaaggga ccagatggaa aagccatcgt ggaatttttg tccaatcttg ctcgtcatac
      901 caaagccgtc aatgttgtgc gtttttctcc aactggggaa attttagcat cgggaggaga
      961 tgatgctgtc atcctattgt ggaaggtgaa tgataacaag gagccggagc agatcgcttt
     1021 tcaggatgag gacgaggccc agctgaacaa ggagaactgg acggttgtga agactctgcg
     1081 gggccactta gaagatgtgt atgatatttg ctgggcaact gatgggaatt taatggcttc
     1141 tgcctctgtg gataacacag ccatcatatg ggatgtcagc aaaggacaaa agatatcaat
     1201 ttttaatgaa cataaaagtt atgtccaagg agtaacctgg gaccctttgg gtcaatatgt
     1261 tgctactctg agctgtgaca gggtgctgcg agtatacagt atacagaaga agcgtgtggc
     1321 tttcaatgtt tcgaagatgc tgtctggaat aggggctgaa ggagaggcaa gaagctaccg
     1381 gatgtttcac gacgacagca tgaagtcttt cttccgtaga ctgagtttca ctcccgacgg
     1441 atctttgctt ctcacgccag ctggatgtgt ggaatctggt gaaaatgtaa tgaataccac
     1501 ttatgttttc tccaggaaga atcttaaaag gcccatcgct catcttccat gtcctggaaa
     1561 agccactctt gctgttcgct gctgtccggt ctactttgaa ctgaggccag tggtggaaac
     1621 gggtgtggag ctgatgagtc tgccctaccg cctggtgttt gctgtggcct cggaggattc
     1681 cgtgcttctg tatgacaccc agcagtcctt cccttttggt tacgtgtcta atatacatta
     1741 ccacaccctc agtgacattt catggtccag cgatggtgcc ttcctggcca tttcttccac
     1801 ggacggttac tgctcatttg tgacatttga gaaagatgaa cttggaattc ctttgaaaga
     1861 gaagccagtt ttgaacatga gaactcctga tacagcaaag aaaaccaaga gtcagacaca
     1921 tcgagggtct tcgccaggac ccagaccggt agagggaacc cctgccagca gaacccaaga
     1981 ccccagcagc cccggcacga ctccccctca ggccagacag gccccagccc caacagtcat
     2041 cagggaccct ccctccatca ctcctgctgt caaaagcccc ttgccggggc cttcggagga
     2101 gaagaccctg cagcccagta gtcaaaacac aaaagcccac ccatcccgga gggtcactct
     2161 gaacacactg caagcctgga gcaagacaac accccggaga ataaacttaa cacccttaaa
     2221 gacggacact ccaccaagtt ctgtaccaac cagtgtgatt tccacccctt ctacagaaga
     2281 aattcagtca gagacgcctg gagacgctca gggcagtccc ccagagctaa agcggcccag
     2341 actcgatgaa aacaaaggag gcacggaaag tctggaccct tagaacccag ctttcttgta
     2401 caaagtggtt gatctagagg gcccgcggtt cgaaggtaag cctatcccta accctctcct
     2461 cggtctcgat tctacgcgta ccggttagta atgagtttaa acgggggagg ctaactgaaa
     2521 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     2581 aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     2641 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     2701 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     2761 ggccctgcca tagcagatct gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt
     2821 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2881 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2941 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3001 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3061 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3121 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3181 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattat
     3241 cctcgagcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag
     3301 cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat
     3361 ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc
     3421 ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa
     3481 tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc
     3541 gggtaggcct ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga
     3601 ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg
     3661 cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc
     3721 tgttctcctc ttcctcatct ccgggccttt cgactctaga cacgtgttga caattaatca
     3781 tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac catggtgagc
     3841 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3901 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3961 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     4021 accttcacct acggcgtgca gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     4081 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     4141 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     4201 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     4261 tacaactaca acagccacaa ggtctatatc accgccgaca agcagaagaa cggcatcaag
     4321 gtgaacttca agacccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     4381 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     4441 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     4501 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggc caagcctttg
     4561 tctcaagaag aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc
     4621 tctgaagact acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt
     4681 gtcaatgtat atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct
     4741 gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc
     4801 atcttgagcc cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa
     4861 gccatagtga aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc
     4921 tctggttatg tgtgggaggg ctaagcactt cgtggccgag gagcaggact gacacgtgct
     4981 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5041 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5101 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5161 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5221 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     5281 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5341 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5401 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5461 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5521 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5581 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5641 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5701 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5761 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5821 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5881 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5941 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6001 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6061 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6121 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6181 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6241 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6301 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6361 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6421 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6481 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6541 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6601 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6661 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6721 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6781 tatggcttca ttcagcttcg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6841 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6901 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6961 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7021 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7081 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7141 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7201 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7261 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7321 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7381 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     7441 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7501 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     7561 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     7621 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gtt
//
