LOCUS       sequence-416575-        7331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-CCM2 lacking amino acids 410-444 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7331)
  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 7331)
  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_d410-444_eGFP
FEATURES             Location/Qualifiers
     source          1..7331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(31..135)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(210..229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        401..780
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        781..984
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     935..955
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1029..1048
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1029..1047
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1089..1802
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(1089..1091,1092..1802)
                     /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(1131..1152)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1392..1411)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1739..1760
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3088..3105)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3094..3318
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3364..3792
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3451..3470)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3661..3682
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3801..3821)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3806..4135
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3986..4121
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4048..4067
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4202..4996
                     /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(4256..4275)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4866..4885
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5170..5291
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5207..5226)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5261..5280
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5340..5356)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5340..5356)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5353..5375)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5364..5380
                     /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(5388..5394,5395..5412,5413..5418))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5433..5454
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5571..5588)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5742..6330)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5822..5841)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6501..7292,7293..7331,1..30))
                     /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"
     primer_bind     7124..7143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
       61 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      121 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
      181 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
      241 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
      301 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
      361 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
      421 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      481 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      541 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      601 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      661 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      721 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      781 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      841 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      901 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      961 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
     1021 atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc
     1081 ttgccaccat gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1141 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1201 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1261 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1321 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1381 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1441 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1501 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1561 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1621 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1681 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1741 tggtcctgct ggagttcgtg accgccgccg ggatcactca cggcatggac gagctgtaca
     1801 agggtaccga gctcggatcc atggaagagg aaggcaaaaa ggggaagaaa cctggaatcg
     1861 tgtccccatt taagagagtg ttcctgaaag gcgagaagag cagggataag aaggcccatg
     1921 aaaaggtcac cgagagaaga cctctgcaca ccgtggtgct gagcctgcct gaaagagtgg
     1981 aacccgatag actgctgagc gactacatcg agaaagaggt gaaatacctg ggccagctga
     2041 catctatccc tggctacctg aaccccagca gccggaccga gatcctgcac ttcatcgaca
     2101 acgccaagcg cgcccaccag cttccaggcc acctgaccca ggagcacgac gccgtgctga
     2161 gcctgagcgc ctacaacgtg aagctggcct ggcgggatgg agaggatatt atcctgagag
     2221 ttcctatcca cgatatcgcc gctgtgtcat atgtgcggga cgatgccgct cacctggtgg
     2281 tcctgaagac agcccaggac cctggcatta gccctagcca gagcctctgc gccgaaagct
     2341 ccagaggcct gtccgcaggc agccttagcg agagcgccgt gggacctgtg gaagcttgtt
     2401 gtctcgtgat cctggccgcc gagagcaagg tcgccgctga agaactgtgc tgcctgctgg
     2461 gccaagtgtt ccaggtggtg tacaccgagt ccactatcga cttcctggac agagccatct
     2521 tcgacggcgc ctctacccct acccaccacc tgagtctgca tagcgacgac agctctacca
     2581 aggtggacat caaggaaacc tacgaggtgg aggccagcac cttctgcttc cccgagagcg
     2641 tggacgtggg cggagcttct ccccacagca agaccatcag cgaatccgag ctgagcgcct
     2701 ctgctacaga actgctgcag gactacatgc tgaccctgag aacaaagctg agcagccagg
     2761 agatccagca gttcgccgcc ctgctgcacg agtacagaaa tggcgctagc atccacgagt
     2821 tctgcatcaa cctgcggcag ctgtacggcg acagcagaaa gttcctgctg ctgggcctgc
     2881 gaccttttat ccccgagaag gacagccaac actttgagaa cttcctggaa acaatcggcg
     2941 tgaaggacgg ccggggaatc atcacagaca gcttcggcag acacagacgg gccctgtcta
     3001 ccacctcctc ctctacaacc aacggcaata gagccacagg ctcttcttaa ctcgagtcta
     3061 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3121 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3181 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3241 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3301 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3361 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3421 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3481 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3541 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3601 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3661 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3721 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3781 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3841 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3901 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3961 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4021 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     4081 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4141 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     4201 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4261 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4321 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4381 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4441 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4501 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4561 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4621 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4681 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4741 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4801 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4861 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4921 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4981 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5041 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5101 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5161 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5221 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5281 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5341 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5401 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5461 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5521 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5581 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5641 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5701 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5761 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5821 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5881 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5941 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6001 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6061 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6121 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6181 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6241 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6301 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6361 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6421 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6481 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6541 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6601 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6661 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6721 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6781 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6841 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6901 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6961 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     7021 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     7081 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     7141 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7201 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7261 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7321 aaaaagggaa t
//
