LOCUS       sequence-416577-        7436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-CCM2 with the W421A and D422A mutations in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7436)
  AUTHORS   Ball NJ, Ghimire S, Follain G, Pajari AO, Wurzinger D, Vaitkeviciute
            M, Cowell AR, Berki B, Ivaska J, Paatero I, Goult BT, Jacquemet G
  TITLE     TLNRD1 is a CCM complex component and regulates endothelial barrier 
            integrity.
  JOURNAL   J Cell Biol. 2024 Sep 2;223(9):e202310030. doi: 
            10.1083/jcb.202310030. Epub 2024 Jul 16.
  PUBMED    39013281
REFERENCE   2  (bases 1 to 7436)
  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: "J Cell 
            Biol."; date: "2024-09-2"; volume: "223(9):e202310030. doi"; pages: 
            " 10.1083/jcb.202310030. Epub 2024 Jul 16"
COMMENT     Sequence Label: CCM2_W421AD422A_eGFP
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..63
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(282..386)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(461..480)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        652..1031
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1032..1235
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1186..1206
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1280..1299
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1280..1298
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1340..2053
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(1340..1342,1343..2053)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1382..1403)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1643..1662)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1990..2011
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3444..3461)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3450..3674
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3720..4148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3807..3826)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4017..4038
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4157..4177)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4162..4491
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4342..4477
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4404..4423
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4558..5352
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4612..4631)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5222..5241
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5526..5647
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5563..5582)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5617..5636
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5696..5712)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5696..5712)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5709..5731)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5720..5736
                     /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(5744..5750,5751..5768,5769..5774))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5789..5810
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5927..5944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6098..6686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6178..6197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6857..7436,1..212,213..281))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
       61 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
      121 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
      181 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
      241 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
      301 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
      361 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
      421 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
      481 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
      541 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      601 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      661 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      721 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      781 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      841 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      901 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      961 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     1021 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     1081 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     1141 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     1201 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
     1261 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt
     1321 taaacttaag cttgccacca tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1381 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1441 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1501 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     1561 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1621 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1681 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1741 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1801 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1861 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1921 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1981 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc acggcatgga
     2041 cgagctgtac aagggtaccg agctcggatc catggaagag gaaggaaaga agggcaagaa
     2101 acctggaatt gtgtctccat ttaagcgggt gtttctgaaa ggcgagaaga gccgggacaa
     2161 gaaagcccac gaaaaagtga ccgagcgccg gcctctgcac accgtcgtgc tgagcctgcc
     2221 tgagagagtc gaacctgaca gactgctgtc cgattacatc gagaaggaag tgaagtacct
     2281 gggccagctg acctccatcc ccggctacct gaatcctagc agcaggaccg aaatcctgca
     2341 tttcatcgac aacgccaagc gggcccacca gctgccagga cacctgactc aggagcacga
     2401 tgccgttctg tctctgagcg cctacaacgt gaagctggcc tggcgggacg gcgaagatat
     2461 catcctgcgg gtgcccatcc acgacatcgc cgctgtgtcc tacgtgcggg acgacgccgc
     2521 ccatctcgtg gtgctgaaga cagcccagga ccccggcatc agccctagcc aaagcctgtg
     2581 cgccgagagc agccggggcc tgagcgccgg cagcctgtcc gaaagcgccg tgggacctgt
     2641 ggaagcttgt tgcctggtca tcctggcagc agaaagcaag gtggccgctg aggaactgtg
     2701 ctgcctgctg ggacaagtgt tccaggtggt gtacaccgag agcaccatcg acttcctgga
     2761 cagagccatc ttcgacggcg cttctacccc tacacaccac ctgagcctgc actctgatga
     2821 tagctccacc aaggtggaca tcaaggagac atacgaggtg gaagcctcta cattctgctt
     2881 ccccgagtct gtggacgtgg gcggcgccag cccccacagc aagaccatca gcgagagcga
     2941 gctgagtgcc tctgccaccg agctgctgca ggactacatg ctgaccctga gaacaaagct
     3001 gtccagccag gagatccagc agttcgccgc cctcctgcac gagtatagaa atggcgcctc
     3061 tatccacgaa ttctgcatca acctgagaca gctgtacggc gattctagaa agttcttact
     3121 gctgggtctt agacctttta tccctgagaa agacagccag cacttcgaga acttcctgga
     3181 gaccatcggc gtgaaggatg gcagaggcat catcacagac agcttcggca gacacagaag
     3241 agccctgtct accaccagca gcagcacgac caacggcaac agagctacag gctctagcga
     3301 cgatagaagc gccccttctg aaggcgacga ggctgctaga atgatcagcg atatcagcag
     3361 cgacattgag gccctgggct gtagcatgga ccaggacagt gcctaactcg agtctagagg
     3421 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3481 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3541 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3601 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3661 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3721 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3781 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3841 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3901 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3961 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4021 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4081 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4141 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4201 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4261 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4321 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4381 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     4441 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     4501 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     4561 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4621 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4681 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4741 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4801 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4861 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4921 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4981 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5041 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     5101 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5161 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5221 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5281 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5341 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5401 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5461 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5521 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5581 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5641 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5701 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5761 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5821 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5881 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5941 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6001 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6061 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6121 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6181 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6241 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6301 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6361 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6421 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6481 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     6541 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6601 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6661 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6721 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6781 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6841 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6901 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6961 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7021 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7081 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7141 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7201 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7261 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7321 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7381 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatag
//
