LOCUS       sequence-349422-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding GET1 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  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 6508)
  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: GET1_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     249..268
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        249..267
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    297..321
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(850..874)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             900..941
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(981..1000)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1025..1073
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1275..1703
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1362..1381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1572..1593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1718..2217
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1753..1776)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2150..2169
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2231..2278
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2297..2299,2300..2302,2303..3013)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2342..2363)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2603..2622)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2950..2971
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3014..3409
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3567..3688
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3604..3623)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3658..3677
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3737..3753)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3737..3753)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3750..3772)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3761..3777
                     /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(3785..3791,3792..3809,3810..3815))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3830..3851
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3968..3985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4139..4727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4219..4238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4898..5689,5690..5758))
                     /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     5521..5540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5759..5863)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5938..5957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6129..6508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
      241 atcgaaatta atacgactca ctatagggag acccaagctg gctagttaag ctatcaacaa
      301 gtttgtacaa aaaagcaggc accatgagct cagccgcggc cgaccactgg gcgtggttgc
      361 tggtgctcag cttcgtgttt ggatgcaatg ttcttaggat cctcctcccg tccttctcat
      421 ccttcatgtc cagggtgctg cagaaggacg cggagcagga gtcacagatg agagcggaga
      481 tccaggacat gaagcaggag ctctccacag tcaacatgat ggacgagttt gccagatatg
      541 ccaggctgga aagaaagatc aacaagatga cggataagct caaaacccat gtgaaagctc
      601 ggacagctca attagccaag ataaaatggg tgataagtgt cgctttctac gtattgcagg
      661 ctgccctgat gatctcactc atttggaagt attattctgt ccctgtggct gtcgtgccga
      721 gtaaatggat aaccccccta gaccgcctgg tagcctttcc tactagagta gcaggtggtg
      781 ttggaattac ctgttggatt ttagtctgta acaaagttgt cgctattgtg cttcatccgt
      841 tcagctgaaa cccagctttc ttgtacaaag tggttgatct agagggcccg cggttcgaag
      901 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gcgtaccggt tagtaatgag
      961 tttaaacggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     1021 atgacggcaa taaaaagaca gaataaaacg cacgggtgtt gggtcgtttg ttcataaacg
     1081 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     1141 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     1201 gctcgcagcc aacgtcgggg cggcaggccc tgccatagca gatctgcgca gctggggctc
     1261 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1321 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1381 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1441 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1501 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1561 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1621 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1681 ttaacaaaaa tttaacgcga attatcctcg agctaccggg taggggaggc gcttttccca
     1741 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
     1801 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
     1861 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
     1921 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
     1981 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
     2041 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
     2101 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     2161 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgact
     2221 ctagacacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa
     2281 ggtgaggaac taaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2341 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2401 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2461 cgtgccctgg cccaccctcg tgaccacctt cacctacggc gtgcagtgct tcgcccgcta
     2521 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2581 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2641 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2701 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaaggtct atatcaccgc
     2761 cgacaagcag aagaacggca tcaaggtgaa cttcaagacc cgccacaaca tcgaggacgg
     2821 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2881 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2941 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3001 cgagctgtac aaggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac
     3061 ggctacaatc aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc
     3121 tagcgacggc cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc
     3181 agaactcgtg gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt
     3241 cgcgatcgga aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct
     3301 tctcgatctg catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc
     3361 agttgggatt cgtgaattgc tgccctctgg ttatgtgtgg gagggctaag cacttcgtgg
     3421 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
     3481 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3541 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3601 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3661 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3721 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3781 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3841 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3901 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3961 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4021 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4081 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4141 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4201 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4261 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4321 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4381 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4441 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4501 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4561 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4621 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4681 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4741 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4801 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4861 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4921 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4981 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5041 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5101 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5161 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5221 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag cttcggttcc caacgatcaa
     5281 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5341 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5401 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5461 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5521 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5581 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5641 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5701 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5761 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5821 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5881 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     5941 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     6001 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     6061 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     6121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     6181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     6241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     6301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     6361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     6421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     6481 ctacgtatta gtcatcgcta ttaccatg
//
