LOCUS       sequence-349721-        6649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding CLDN8 and EmGFP-Bsd.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6649)
  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 6649)
  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: CLDN8_pcDNA6.2/EmGFP-Bsd
FEATURES             Location/Qualifiers
     source          1..6649
                     /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(1003..1027)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1053..1094
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1134..1153)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1178..1226
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1428..1856
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1515..1534)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1725..1746
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1871..2358
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1906..1929)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2291..2310
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2372..2419
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(2438..2440,2441..2443,2444..3154)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2483..2504)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2744..2763)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3091..3112
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3155..3550
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3708..3829
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3745..3764)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3799..3818
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3878..3894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3878..3894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3891..3913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3902..3918
                     /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(3926..3932,3933..3950,3951..3956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3971..3992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4109..4126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4280..4868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4360..4379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5039..5830,5831..5899))
                     /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     5662..5681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5900..6004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6079..6098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6270..6649
                     /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 accatggcaa cccatgcctt agaaatcgct gggctgtttc
      361 ttggtggtgt tggaatggtg ggcacagtgg ctgtcactgt catgcctcag tggagagtgt
      421 cggccttcat tgaaaacaac atcgtggttt ttgaaaactt ctgggaagga ctgtggatga
      481 attgcgtgag gcaggctaac atcaggatgc agtgcaaaat ctatgattcc ctgctggctc
      541 tttctccgga cctacaggca gccagaggac tgatgtgtgc tgcttccgtg atgtccttct
      601 tggctttcat gatggccatc cttggcatga aatgcaccag gtgcacgggg gacaatgaga
      661 aggtgaaggc tcacattctg ctgacggctg gaatcatctt catcatcacg ggcatggtgg
      721 tgctcatccc tgtgagctgg gttgccaatg ccatcatcag agatttctat aactcaatag
      781 tgaatgttgc ccaaaaacgt gagcttggag aagctctcta cttaggatgg accacggcac
      841 tggtgctgat tgttggagga gctctgttct gctgcgtttt ttgttgcaac gaaaagagca
      901 gtagctacag atactcgata ccttcccatc gcacaaccca aaaaagttat cacaccggaa
      961 agaagtcacc gagcgtctac tccagaagtc agtatgtgta gaacccagct ttcttgtaca
     1021 aagtggttga tctagagggc ccgcggttcg aaggtaagcc tatccctaac cctctcctcg
     1081 gtctcgattc tacgcgtacc ggttagtaat gagtttaaac gggggaggct aactgaaaca
     1141 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     1201 acgcacgggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     1261 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     1321 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     1381 ccctgccata gcagatctgc gcagctgggg ctctaggggg tatccccacg cgccctgtag
     1441 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1501 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1561 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1621 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1681 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1741 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1801 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattatcc
     1861 tcgagctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     1921 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     1981 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     2041 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     2101 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     2161 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     2221 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     2281 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     2341 tcatctccgg gcctttcgac tctagacacg tgttgacaat taatcatcgg catagtatat
     2401 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gtgagcaagg gcgaggagct
     2461 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2521 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2581 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcacctacgg
     2641 cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2701 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2761 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2821 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2881 ccacaaggtc tatatcaccg ccgacaagca gaagaacggc atcaaggtga acttcaagac
     2941 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     3001 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     3061 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     3121 cgggatcact ctcggcatgg acgagctgta caaggccaag cctttgtctc aagaagaatc
     3181 caccctcatt gaaagagcaa cggctacaat caacagcatc cccatctctg aagactacag
     3241 cgtcgccagc gcagctctct ctagcgacgg ccgcatcttc actggtgtca atgtatatca
     3301 ttttactggg ggaccttgtg cagaactcgt ggtgctgggc actgctgctg ctgcggcagc
     3361 tggcaacctg acttgtatcg tcgcgatcgg aaatgagaac aggggcatct tgagcccctg
     3421 cggacggtgc cgacaggtgc ttctcgatct gcatcctggg atcaaagcca tagtgaagga
     3481 cagtgatgga cagccgacgg cagttgggat tcgtgaattg ctgccctctg gttatgtgtg
     3541 ggagggctaa gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
     3601 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3661 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3721 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3781 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3841 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3901 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3961 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5401 gcttcggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5641 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5701 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5761 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5821 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5881 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5941 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6001 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6061 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6121 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6181 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     6241 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     6301 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6361 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6421 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6481 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     6541 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     6601 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatg
//
