LOCUS       sequence-349731-        6685 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding MIS18A and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6685)
  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 6685)
  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: MIS18A_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6685
                     /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(1407..1431)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1457..1498
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1538..1557)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1582..1630
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1832..2260
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1919..1938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2129..2150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2275..2774
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2310..2333)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2707..2726
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2788..2835
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2854..2856,2857..2859,2860..3570)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2899..2920)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3160..3179)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3507..3528
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3571..3966
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4124..4245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4161..4180)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4215..4234
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4294..4310)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4294..4310)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4307..4329)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4318..4334
                     /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(4342..4348,4349..4366,4367..4372))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4387..4408
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4525..4542)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4696..5284)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4776..4795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5455..6246,6247..6315))
                     /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     6078..6097
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6316..6420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6495..6514)
                     /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 accatggcag gcgttcggtc
      721 actgaggtgt agcagaggat gcgctggcgg ctgtgagtgc ggcgacaagg gcaaatgcag
      781 cgactcctcg ctgttgggca agagactctc cgaagactcg agccgccacc agctgttgca
      841 gaagtgggcg agcatgtgga gctccatgag cgaagacgcg tcggtggccg acatggagag
      901 ggcgcagctg gaggaggagg cggcggctgc ggaggagagg ccgctggtgt tcctgtgctc
      961 cggctgccgg cggccgctgg gcgactcgct gagctgggtg gccagccagg aggacaccaa
     1021 ctgcatcctg cttcgctgtg tttcctgtaa tgtttctgtg gataaggaac agaagctatc
     1081 caaacgtgaa aaggaaaatg gttgcgtcct tgagactttg tgctgcgcgg ggtgctcact
     1141 caatcttggc tacgtgtaca gatgcacgcc caagaatctt gattacaaga gagacttgtt
     1201 ttgcctcagt gttgaagcca ttgaaagtta tgttttaggg tcctctgaaa agcaaattgt
     1261 gtcagaagat aaagagcttt ttaatcttga aagcagagtt gaaatagaaa agtctctaac
     1321 acagatggaa gatgtcttga aagcattaca aatgaagctg tgggaggccg aatccaaatt
     1381 gtcctttgcc acttgtaaaa gctagaaccc agctttcttg tacaaagtgg ttgatctaga
     1441 gggcccgcgg ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg
     1501 taccggttag taatgagttt aaacggggga ggctaactga aacacggaag gagacaatac
     1561 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg
     1621 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     1681 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     1741 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcagat
     1801 ctgcgcagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     1861 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1921 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1981 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2041 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2101 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2161 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2221 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt atcctcgagc taccgggtag
     2281 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     2341 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     2401 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     2461 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     2521 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     2581 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     2641 ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag
     2701 gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat
     2761 ctccgggcct ttcgactcta gacacgtgtt gacaattaat catcggcata gtatatcggc
     2821 atagtataat acgacaaggt gaggaactaa accatggtga gcaagggcga ggagctgttc
     2881 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2941 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3001 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac ctacggcgtg
     3061 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3121 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3181 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3241 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3301 aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc
     3361 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3421 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3481 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3541 atcactctcg gcatggacga gctgtacaag gccaagcctt tgtctcaaga agaatccacc
     3601 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     3661 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     3721 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     3781 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     3841 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     3901 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
     3961 ggctaagcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
     4021 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4081 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4141 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4201 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4261 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4321 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4381 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4441 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4501 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4561 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4621 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4681 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4741 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4801 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4861 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4921 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4981 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5041 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5101 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5161 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5221 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5281 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5341 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5401 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5461 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5521 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5581 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     5641 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5701 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5761 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctt
     5821 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5881 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5941 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6001 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6061 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6121 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6181 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6241 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6301 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6361 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6421 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6481 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6541 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6601 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     6661 gatgtacggg ccagatatac gcgtt
//
