LOCUS       sequence-416572-        7436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-CCM2 with the I428S mutation  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_I428S_eGFP
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     172..191
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(410..514)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(589..608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        780..1159
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1160..1363
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1314..1334
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1408..1427
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1408..1426
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1468..2181
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(1468..1470,1471..2181)
                     /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(1510..1531)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1771..1790)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2118..2139
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3572..3589)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3578..3802
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3848..4276
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3935..3954)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4145..4166
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4285..4305)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4290..4619
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4470..4605
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4532..4551
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4686..5480
                     /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(4740..4759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5350..5369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5654..5775
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5691..5710)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5745..5764
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5824..5840)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5824..5840)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5837..5859)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5848..5864
                     /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(5872..5878,5879..5896,5897..5902))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5917..5938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6055..6072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6226..6814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6306..6325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6985..7436,1..340,341..409))
                     /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 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
       61 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
      121 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
      181 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
      241 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
      301 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
      361 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
      421 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
      481 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
      541 acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc
      601 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta
      661 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa
      721 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg
      781 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
      841 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      901 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
      961 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1021 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1081 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1141 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1201 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1261 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1321 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga
     1381 acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg
     1441 ctagcgttta aacttaagct tgccaccatg agcaagggcg aggagctgtt caccggggtg
     1501 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1561 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1621 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     1681 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1741 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1801 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1861 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     1921 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     1981 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2041 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     2101 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactcac
     2161 ggcatggacg agctgtacaa gggtaccgag ctcggatcca tggaagagga aggcaagaaa
     2221 ggcaagaagc ctggaattgt gtctcctttt aagagagttt tcctgaaggg agagaaaagc
     2281 agagacaaga aggcccacga aaaggtgacc gagagaagac ctctgcacac cgtggtgctg
     2341 tctctgcctg aacgggtgga accagacaga ctgctcagtg attatatcga gaaggaagtg
     2401 aagtacctgg gccaactgac cagcatccct ggctacctga acccctctag cagaacagaa
     2461 atcctgcact ttattgataa tgccaagaga gcccaccagc tgcccggcca cctgacccag
     2521 gagcacgatg ccgtcctgtc tctgagcgcc tacaacgtga agctggcctg gcgggatggc
     2581 gaggacatca tcctcagagt gcccatccac gacatcgccg ccgtgtccta cgtgcgggac
     2641 gacgccgctc atctggtggt gctgaagacc gcccaggacc ctggcatcag ccccagccag
     2701 agcctgtgcg ccgagagctc ccggggcctg agcgcaggct ctctgtctga aagcgccgtg
     2761 ggtcctgttg aggcatgctg cctggtcatc ctggctgccg agtcaaaagt ggccgctgag
     2821 gagctgtgtt gcctgctggg ccaggtgttt caggtggtgt acaccgagag caccatcgac
     2881 ttcctggaca gagctatctt cgacggcgct agcaccccta cccaccacct ctccctgcat
     2941 tctgatgaca gctccacaaa ggtggacatc aaggagacat acgaggtgga agccagcaca
     3001 ttctgcttcc ccgagagcgt cgatgtgggc ggcgccagcc cccactctaa aacaatcagc
     3061 gaaagcgagc tgagcgccag cgccacagag ctgctgcagg actacatgct gaccctgaga
     3121 accaagctga gcagccagga gatccagcag ttcgccgccc tgctgcacga gtacagaaat
     3181 ggcgccagca tccacgagtt ctgcatcaac ctgaggcagc tgtacggcga cagcagaaag
     3241 ttcctgctgc tgggactgcg gcctttcatc ccagaaaagg acagccaaca cttcgagaac
     3301 ttcctggaaa ccatcggcgt gaaagacgga agaggcatca tcaccgacag cttcggcaga
     3361 caccggcggg ccctgagcac cacctctagc agcactacaa acggcaacag agctacaggc
     3421 agcagcgatg atcgcagtgc cccttctgag ggagatgaat gggaccggat gatcagcgac
     3481 tctagcagcg acattgaggc cctgggctgt agcatggacc aggactccgc ctaactcgag
     3541 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     3601 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3661 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3721 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3781 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     3841 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     3901 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     3961 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4021 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4081 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4141 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4201 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4261 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4321 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     4381 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4441 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4501 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4561 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4621 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     4681 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     4741 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4801 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4861 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4921 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4981 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     5041 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5101 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5161 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     5221 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5281 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     5341 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     5401 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     5461 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     5521 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5581 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     5641 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     5701 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     5761 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     5821 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5881 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5941 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6001 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6661 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6781 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     6841 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     6901 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6961 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7021 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7081 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7141 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7201 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7261 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7321 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7381 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtca
//
