LOCUS       sequence-349693-        6460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding TMEM50B and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  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 6460)
  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: TMEM50B_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6460
                     /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(1182..1206)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1232..1273
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1313..1332)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1357..1405
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1607..2035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1694..1713)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1904..1925
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2050..2549
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2085..2108)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2482..2501
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2563..2610
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2629..2631,2632..2634,2635..3345)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2674..2695)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2935..2954)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3282..3303
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3346..3741
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3899..4020
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3936..3955)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3990..4009
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4069..4085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4069..4085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4082..4104)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4093..4109
                     /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(4117..4123,4124..4141,4142..4147))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4162..4183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4300..4317)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4471..5059)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4551..4570)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5230..6021,6022..6090))
                     /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     5853..5872
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6091..6195)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6270..6289)
                     /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 gcttcctaga
      721 taattttcgt tggccagaat gtgaatgtat tgactggagt gagagaagaa atgctgtggc
      781 atctgttgtc gcaggtatat tgttttttac aggctggtgg ataatgattg atgcagctgt
      841 ggtgtatcct aagccagaac agttgaacca tgcctttcac acatgtggtg tattttccac
      901 attggctttc ttcatgataa atgctgtatc caatgctcag gtgagaggtg atagctatga
      961 aagcggctgt ttaggaagaa caggtgctcg agtttggctt ttcattggtt tcatgttgat
     1021 gtttgggtca cttattgctt ccatgtggat tctttttggt gcatatgtta cccaaaatac
     1081 tgatgtttat ccgggactag ctgtgttttt tcaaaatgca cttatatttt ttagcactct
     1141 gatctacaaa tttggaagaa ccgaagagct atggacctag aacccagctt tcttgtacaa
     1201 agtggttgat ctagagggcc cgcggttcga aggtaagcct atccctaacc ctctcctcgg
     1261 tctcgattct acgcgtaccg gttagtaatg agtttaaacg ggggaggcta actgaaacac
     1321 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     1381 cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     1441 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     1501 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     1561 cctgccatag cagatctgcg cagctggggc tctagggggt atccccacgc gccctgtagc
     1621 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1681 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1741 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1801 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1861 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1921 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1981 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattatcct
     2041 cgagctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag
     2101 ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca
     2161 ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc
     2221 ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg
     2281 aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg
     2341 taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc
     2401 tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc
     2461 gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt
     2521 tctcctcttc ctcatctccg ggcctttcga ctctagacac gtgttgacaa ttaatcatcg
     2581 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggtgagcaag
     2641 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2701 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2761 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2821 ttcacctacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     2881 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2941 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3001 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3061 aactacaaca gccacaaggt ctatatcacc gccgacaagc agaagaacgg catcaaggtg
     3121 aacttcaaga cccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3181 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     3241 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3301 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggccaa gcctttgtct
     3361 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     3421 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     3481 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     3541 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     3601 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     3661 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     3721 ggttatgtgt gggagggcta agcacttcgt ggccgaggag caggactgac acgtgctacg
     3781 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3841 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3901 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3961 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4021 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4081 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4141 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4201 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4261 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4321 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4381 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4441 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4501 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4561 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4621 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4681 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4741 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4801 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4861 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4921 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4981 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5041 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5101 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5161 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5221 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5281 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5341 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5401 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5461 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5521 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5581 ggcttcattc agcttcggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5641 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5701 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5761 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5821 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5881 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5941 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6001 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6061 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6121 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6181 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6241 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6301 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     6361 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     6421 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
//
