LOCUS       sequence-350165-        6618 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding Rcan1 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6618)
  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 6618)
  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: Rcan1_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6618
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1574..1598)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1624..1665
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1705..1724)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1749..1797
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1999..2427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2086..2105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2296..2317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2442..2941
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2477..2500)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2874..2893
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2955..3002
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3021..3023,3024..3026,3027..3737)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3066..3087)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3327..3346)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3674..3695
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3738..4133
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4291..4412
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4328..4347)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4382..4401
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4461..4477)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4461..4477)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4474..4496)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4485..4501
                     /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(4509..4515,4516..4533,4534..4539))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4554..4575
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4692..4709)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4863..5451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4943..4962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5622..6413,6414..6482))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRXDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPXLNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6245..6264
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6483..6587)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggctccgcg gccgccccct tcaccatgca
      961 ttttagggac tttagctaca attttagctc cctgattgct tgtgtggcaa acgatgatgt
     1021 cttcagcgaa agtgagacca gggccaaatt tgaatccctc ttcagaacat atgacaagga
     1081 caccaccttc cagtatttta agagcttcaa acgtgtccgg ataaacttca gcaacccctt
     1141 atctgcagcc gatgccaggc tgcggctgca caagaccgag ttcctgggga aggaaatgaa
     1201 gttgtatttt gctcagactt tacacatagg aagttcacac ctggctccgc ccaatcccga
     1261 caaacagttc ctcatctccc ctccggcctc tcctcccgtt ggctggaaac aagtagaaga
     1321 tgccaccccc gtcataaatt acgatctttt atatgccatc tccaagctgg ggccaggaga
     1381 gaagtatgaa ctgcatgcag cgacagacac cactcccagt gtggtggtcc acgtgtgtga
     1441 gagtgaccaa gagaatgagg aggaagagga agagatggag agaatgaaga gacccaagcc
     1501 caaaatcatc cagacacgga gaccggagta cacacccatc cacctcagct gaaccggaag
     1561 ggtgggcgcg ccgacccagc tttcttgtac aaagtggttg atctagaggg cccgcggttc
     1621 gaaggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggttagtaa
     1681 tgagtttaaa cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
     1741 cgctatgacg gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata
     1801 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     1861 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     1921 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcagatctg cgcagctggg
     1981 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2041 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2101 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     2161 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     2221 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     2281 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     2341 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     2401 tgatttaaca aaaatttaac gcgaattatc ctcgagctac cgggtagggg aggcgctttt
     2461 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     2521 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     2581 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     2641 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     2701 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     2761 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     2821 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     2881 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc
     2941 gactctagac acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     3001 acaaggtgag gaactaaacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     3061 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     3121 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     3181 tgcccgtgcc ctggcccacc ctcgtgacca ccttcaccta cggcgtgcag tgcttcgccc
     3241 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     3301 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     3361 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     3421 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaag gtctatatca
     3481 ccgccgacaa gcagaagaac ggcatcaagg tgaacttcaa gacccgccac aacatcgagg
     3541 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     3601 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     3661 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     3721 tggacgagct gtacaaggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
     3781 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     3841 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     3901 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     3961 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
     4021 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
     4081 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc
     4141 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat
     4201 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4261 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4321 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4381 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4441 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4501 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4561 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4621 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4681 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4741 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4801 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4861 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4921 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4981 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5041 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5101 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5161 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5221 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5281 cctaactacg gctacactag aagracagta tttggtatct gcgctctgct gaagccagtt
     5341 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5401 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5461 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5521 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5581 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5641 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5701 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5761 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5821 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5881 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5941 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctycgg ttcccaacga
     6001 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6061 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6121 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6181 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaya
     6241 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6301 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6361 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6421 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6481 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6541 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6601 aaagtgccac ctgacgtc
//
