LOCUS       sequence-349016-        6804 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression vector encoding LRRC3 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6804)
  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 6804)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: LRRC3_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6804
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(180..199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        371..750
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        751..954
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     905..925
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     999..1018
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        999..1017
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1047..1071
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1895..1919)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1945..1986
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2026..2045)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2070..2118
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2320..2748
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2407..2426)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2617..2638
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2763..3263
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2798..2821)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3196..3215
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3277..3324
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3343..3345,3346..3348,3349..4059)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3388..3409)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3649..3668)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3996..4017
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4060..4455
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4613..4734
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4650..4669)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4704..4723
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4783..4799)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4783..4799)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4796..4818)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4807..4823
                     /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(4831..4837,4838..4855,4856..4861))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4876..4897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5014..5031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5185..5773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5265..5284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5944..6735,6736..6804))
                     /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     6567..6586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt
      181 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
      241 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
      301 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
      361 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      421 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      481 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      541 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      601 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      661 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      721 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      781 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      841 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      901 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
      961 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     1021 acccaagctg gctagttaag ctatcaacaa gtttgtacaa aaaagcaggc tccgcggccg
     1081 cccccttcgc gcccaccctt caccatgggc accgtgcgcc cacctcgccc ctcgctcctg
     1141 ctggtctcca cccgggagtc ttgtctcttc ctcctcttct gcctgcacct gggcgccgcc
     1201 tgcccacagc cctgccggtg ccctgaccac gcaggggctg tggctgtctt ctgcagcttg
     1261 cggggccttc aggaggtccc cgaggacatc ccggccaaca ccgtgctcct gaagctcgat
     1321 gccaacaaga tctcccacct cccggacggg gccttccagc acctgcaccg gctcagggag
     1381 ctggatctgt ctcacaacgc catcgaggcc atcggctccg ccaccttcgc gggcctggcc
     1441 gggggcctgc ggctgctgga cctgtcttac aaccgcatcc agaggatccc caaggacgcc
     1501 ctgggcaaac tcagcgccaa gatacgcctg tcccacaacc ccctgcactg cgagtgcgcc
     1561 ctgcaggagg ccctgtggga gctgaagctg gaccccgact ctgtggacga gatcgcctgc
     1621 cacacctcag tgcaggagga gtttgtgggg aagcctctgg ttcaggctct ggatgcgggt
     1681 gccagcctct gcagcgtccc ccacaggacc acagacgtgg ccatgctggt caccatgttc
     1741 ggctggttcg ccatggtgat cgcctacgtc gtgtactatg tgcgccacaa ccaggaggat
     1801 gcccggaggc acctggagta cctgaagtct ctgcccagcg cccccgcctc caaggacccc
     1861 atcggcccgg ggccctagaa gggtgggcgc gccgacccag ctttcttgta caaagtggtt
     1921 gatctagagg gcccgcggtt cgaaggtaag cctatcccta accctctcct cggtctcgat
     1981 tctacgcgta ccggttagta atgagtttaa acgggggagg ctaactgaaa cacggaagga
     2041 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     2101 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     2161 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     2221 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     2281 tagcagatct gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2341 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2401 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2461 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2521 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2581 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2641 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     2701 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattat cctcgagcta
     2761 ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct
     2821 gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag
     2881 gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt
     2941 caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc
     3001 acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct
     3061 ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag
     3121 gggtgggtcc ggggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt
     3181 cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct
     3241 cttcctcatc tccgggcctt tcgactctag acacgtgttg acaattaatc atcggcatag
     3301 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggtgag caagggcgag
     3361 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3421 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     3481 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcacc
     3541 tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     3601 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     3661 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     3721 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     3781 aacagccaca aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc
     3841 aagacccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3901 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     3961 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     4021 gccgccggga tcactctcgg catggacgag ctgtacaagg ccaagccttt gtctcaagaa
     4081 gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac
     4141 tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta
     4201 tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg
     4261 gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc
     4321 ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg
     4381 aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat
     4441 gtgtgggagg gctaagcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
     4501 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4561 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4621 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4681 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4741 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4801 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4861 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4921 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4981 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5041 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5101 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5161 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5221 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5281 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5341 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5401 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5461 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5521 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5581 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5641 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5701 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5761 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5821 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5881 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5941 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6001 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6061 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6121 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6181 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6241 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6301 attcagcttc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6361 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6421 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6481 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6541 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6601 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6661 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6721 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6781 gacacggaaa tgttgaatac tcat
//
