LOCUS       sequence-349423-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding RRP1 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  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 7369)
  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: RRP1_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..7369
                     /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(1711..1735)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1761..1802
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1842..1861)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1886..1934
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2136..2564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2223..2242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2433..2454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2579..3078
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2614..2637)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3011..3030
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3092..3139
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3158..3160,3161..3163,3164..3874)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3203..3224)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3464..3483)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3811..3832
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3875..4270
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4428..4549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4465..4484)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4519..4538
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4598..4614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4598..4614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4611..4633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4622..4638
                     /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(4646..4652,4653..4670,4671..4676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4691..4712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4829..4846)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5000..5588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5080..5099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5759..6550,6551..6619))
                     /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     6382..6401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6620..6724)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6799..6818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6990..7369
                     /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 accatggttt cgcgcgtgca gctcccgcct gagatccagc
      361 tggctcagcg cctggcgggg aatgagcagg tgacccggga ccgggcggtg aggaagctcc
      421 ggaaatacat cgtcgccagg actcagcggg ccgcaggtgg ttttacgcac gacgagctgc
      481 tgaaggtgtg gaaaggactg ttttattgca tgtggatgca ggacaagcca ctcctccagg
      541 aagaattagg aaggactatt tcccagctcg ttcatgcttt tcagaccacg gaggcgcagc
      601 acctgttcct tcaggccttc tggcagacca tgaatcgcga gtggacgggc attgacaggc
      661 tgcgcctgga taaattctac atgctcatgc ggatggtcct gaacgagtcc ttgaaggttc
      721 tgaagatgca aggctgggaa gaaagacaga tcgaggagct gctagagctg ctgatgactg
      781 agatcctgca ccccagcagc caggccccca acggtgtgaa gagccacttc atcgagatct
      841 tcctggagga gctgaccaaa gtgggcgccg aggagcttac ggcagaccag aacctgaagt
      901 tcatcgaccc cttctgcaga atcgctgccc ggaccaagga ttccttggtt ttgaacaaca
      961 tcactcgagg catctttgag acgattgtgg agcaggcccc gcttgccatt gaagacctcc
     1021 tgaatgaact ggacacacag gatgaggagg tggcgtcgga cagtgatgag tcctctgagg
     1081 gtggtgagcg tggagacgcg ctgtcccaga agaggtctga gaagccgccc gcaggctcca
     1141 tctgcagggc tgaacctgag gctggtgagg agcaggcagg tgacgacagg gacagtggcg
     1201 gccccgttct ccagtttgac tacgaggcag ttgctaacag actgtttgaa atggccagcc
     1261 gccagagcac cccttctcag aacagaaagc gtctctacaa agtgatccgg aagctgcagg
     1321 acctggcagg aggcattttc cctgaagatg agatcccaga gaaggcctgc aggcgcctgc
     1381 ttgaagggag gcggcagaag aagacgaaga agcagaagcg tctgctcagg ttgcagcagg
     1441 agagagggaa aggtgagaag gagcccccga gcccgggcat ggagaggaag aggagcagga
     1501 ggaggggtgt aggggccgac cccgaggcgc gggcagaggc tggtgagcag ccaggcacag
     1561 ctgagcgggc cctgctccga gatcagccca ggggccgtgg ccagagaggg gctcgccaga
     1621 gaaggaggac acctcggccc ctgaccagtg cccgagcaaa ggcggccaat gtccaggagc
     1681 cggagaagaa gaagaaacgc agggagtaga acccagcttt cttgtacaaa gtggttgatc
     1741 tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta
     1801 cgcgtaccgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg gaaggagaca
     1861 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacgggtgt
     1921 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     1981 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     2041 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     2101 agatctgcgc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     2161 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2221 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2281 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2341 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2401 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2461 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2521 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattatcctc gagctaccgg
     2581 gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc
     2641 acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc
     2701 caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg
     2761 aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt
     2821 ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg
     2881 ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt
     2941 gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     3001 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     3061 tcatctccgg gcctttcgac tctagacacg tgttgacaat taatcatcgg catagtatat
     3121 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gtgagcaagg gcgaggagct
     3181 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     3241 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     3301 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg
     3361 cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     3421 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     3481 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     3541 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     3601 ccacaaggtc tatatcaccg ccgacaagca gaagaacggc atcaaggtga acttcaagac
     3661 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     3721 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     3781 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3841 cgggatcact ctcggcatgg acgagctgta caaggccaag cctttgtctc aagaagaatc
     3901 caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag
     3961 cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca
     4021 ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc
     4081 tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg
     4141 cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga
     4201 cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg
     4261 ggagggctaa gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
     4321 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4381 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4441 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4501 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4561 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4621 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4681 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4741 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4801 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4861 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4921 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5161 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5401 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     5461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6061 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6121 gcttcggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6361 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6721 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6781 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6841 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6901 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     6961 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     7021 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     7081 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     7141 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     7201 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     7261 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     7321 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatg
//
