LOCUS       sequence-349715-        6914 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding PCBP3 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6914)
  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 6914)
  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: PCBP3_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6914
                     /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(1647..1671)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1697..1738
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1778..1797)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1822..1870
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2072..2500
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2159..2178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2369..2390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2515..3003
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2550..2573)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2936..2955
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        3017..3064
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(3083..3085,3086..3088,3089..3799)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3128..3149)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3389..3408)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3736..3757
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3800..4195
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4353..4474
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4390..4409)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4444..4463
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4523..4539)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4523..4539)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4536..4558)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4547..4563
                     /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(4571..4577,4578..4595,4596..4601))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4616..4637
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4754..4771)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4925..5513)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5005..5024)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5684..6475,6476..6544))
                     /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     6307..6326
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6545..6649)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6724..6743)
                     /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 accatggagt ccaaggtctc
      721 agaaggtggc ctgaatgtga ccctcaccat ccgcctgctg atgcatggaa aggaagttgg
      781 aagcatcatc gggaagaaag gagaaactgt gaagaagatg cgtgaggaga gtggtgcaag
      841 gatcaacatc tcagagggaa actgcccaga gaggattgtg accatcacag gccccacaga
      901 cgccatcttc aaggcctttg ccatgatcgc atacaagttt gaggaggata tcatcaactc
      961 catgagcaac agccctgcca ccagcaagcc cccagtgacg ctgaggctgg tggtgcctgc
     1021 cagccagtgc gggtccctga tcggcaaagg aggctccaag atcaaggaga tcagggagtc
     1081 cacaggtgcc caggtgcagg tggctgggga catgctgccc aactccacgg agcgagcggt
     1141 gaccatctcg gggaccccag atgccatcat ccagtgcgtc aagcagatct gtgtggtcat
     1201 gctggaggcc tacacaatcc agggacagta tgccatccct cacccggatt tgaccaagct
     1261 ccaccagttg gccatgcagc aaaccccctt tcctcccctc ggacagacca accccgcttt
     1321 ccccggagaa aagctgcctt tacactcctc cgaagaagct caaaatctga tgggccagtc
     1381 atcaggtctg gacgccagcc caccggccag cactcatgag ctcaccattc ccaatgatct
     1441 aataggctgc ataattggac gccaagggac caaaatcaat gaaattcgac agatgtctgg
     1501 agctcagatc aaaatcgcca acgccacgga agggtcctca gagcgtcaga tcaccatcac
     1561 ggggaccccg gccaacatca gccttgccca gtatctcatc aacgccaggc tgacgtccga
     1621 ggtcaccggg atgggcacgc tgtagaaccc agctttcttg tacaaagtgg ttgatctaga
     1681 gggcccgcgg ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg
     1741 taccggttag taatgagttt aaacggggga ggctaactga aacacggaag gagacaatac
     1801 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg
     1861 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     1921 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     1981 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcagat
     2041 ctgcgcagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     2101 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2161 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2221 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2281 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2341 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2401 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2461 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt atcctcgagc taccgggtag
     2521 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     2581 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     2641 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     2701 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     2761 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     2821 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggg
     2881 ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg cccggcattc
     2941 tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc tccgggcctt
     3001 tcgactctag acacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
     3061 cgacaaggtg aggaactaaa ccatggtgag caagggcgag gagctgttca ccggggtggt
     3121 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     3181 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3241 gctgcccgtg ccctggccca ccctcgtgac caccttcacc tacggcgtgc agtgcttcgc
     3301 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3361 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3421 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3481 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca aggtctatat
     3541 caccgccgac aagcagaaga acggcatcaa ggtgaacttc aagacccgcc acaacatcga
     3601 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3661 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3721 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3781 catggacgag ctgtacaagg ccaagccttt gtctcaagaa gaatccaccc tcattgaaag
     3841 agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg ccagcgcagc
     3901 tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta ctgggggacc
     3961 ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca acctgacttg
     4021 tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac ggtgccgaca
     4081 ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg atggacagcc
     4141 gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg gctaagcact
     4201 tcgtggccga ggagcaggac tgacacgtgc tacgagattt cgattccacc gccgccttct
     4261 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4321 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4381 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4441 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4501 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4561 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4621 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4681 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4741 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4801 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4861 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4921 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4981 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5041 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5101 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5161 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5221 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5281 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5341 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5401 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5461 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5521 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5581 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5641 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5701 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5761 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5821 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5881 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5941 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6001 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagcttc ggttcccaac
     6061 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6121 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6181 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6241 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6301 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6361 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6421 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6481 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6541 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6601 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6661 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6721 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6781 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     6841 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     6901 cagatatacg cgtt
//
