LOCUS       sequence-416570-        7436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-CCM2 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_wt_eGFP
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(23..91,92..883)
                     /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     complement(241..260)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1054..1642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1543..1562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1796..1813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1930..1951
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1966..1971,1972..1989,1990..1996)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2004..2020
                     /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)."
     primer_bind     2009..2031
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2028..2044
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2028..2044
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2093..2214
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2104..2123)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2158..2177
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2388..3182)
                     /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(2499..2518)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3109..3128
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3249..3578)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3263..3398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3317..3336)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3563..3583
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3592..4020)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3702..3723)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3914..3933
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4066..4290
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4279..4296
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5687..6400)
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(join(5687..6397,6398..6400))
                     /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(5729..5750)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6078..6097
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6337..6358
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6441..6460)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6442..6460)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6505..6708)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6534..6554)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6709..7088
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     7260..7279
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7354..7436,1..22)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
       61 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
      121 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
      181 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
      241 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
      301 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
      361 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
      421 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
      481 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
      541 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
      601 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
      661 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
      721 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
      781 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
      841 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
      901 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
      961 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1021 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1081 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1141 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1201 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1261 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1321 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1381 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1441 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1501 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1561 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1621 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     1681 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     1741 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     1801 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     1861 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     1921 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     1981 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     2041 tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac
     2101 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     2161 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg
     2221 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     2281 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     2341 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg
     2401 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     2461 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     2521 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     2581 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     2641 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     2701 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     2761 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     2821 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     2881 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     2941 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     3001 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     3061 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     3121 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     3181 atgcgaaacg atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc
     3241 gggagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     3301 tcagaggcag aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     3361 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     3421 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     3481 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     3541 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt
     3601 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3661 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3721 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3781 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3841 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3901 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3961 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     4021 ggggataccc cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg
     4081 catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc
     4141 caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt
     4201 aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa
     4261 caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaacgggcc
     4321 ctctagactc gagttaggca gagtcctggt ccatgctaca tcccagggcc tcgatgtcgc
     4381 tgctgatatc gctgatcatc cggtcccact catctccctc gctaggggcg cttctatcgt
     4441 cgctagagcc tgtggctctg ttgccgttgg ttgtagaaga gcttgtggta ctcagggctc
     4501 ttctgtgtct gccgaagctg tctgttatga tgccccgtcc gtccttcact ccgatggttt
     4561 ccaggaagtt ctcgaagtgc tgggagtcct tctcagggat gaatggccgc aggcccagca
     4621 gaaggaactt acggctatcg ccgtacagct gtctcaggtt gatgcagaac tcgtggatgc
     4681 tagcgccatt tctgtactcg tggagcagag cggcgaactg ctgaatctcc tggctgctca
     4741 gcttggttct cagggtcagc atataatcct gcaacagctc cgtggcgctg gctgacagtt
     4801 cagactcgct gatggtcttg ctgtgagggc tggcgccgcc cacgtcgacg ctttcgggga
     4861 agcagaaggt gctggcttcc acctcgtagg tttccttaat gtccaccttt gtgctgctgt
     4921 catcgctatg caggctcagg tggtgggtag gggtagaggc gccgtcgaag atggctctgt
     4981 ccaggaagtc gattgtgctc tcggtgtaca cgacctgaaa cacctggccc agcagacagc
     5041 agagctcttc agcggccacc ttgctctcgg cggccaggat caccaggcag catgcttcca
     5101 caggtccaac agcggattcg ctcaggctgc cggcggacag gccgcgggag gactcggcgc
     5161 acagagattg gcttggggag atgccggggt cctgggcggt tttcagcacc accaggtgag
     5221 cggcatcatc ccgcacgtag gacacggcgg cgatatcgtg gatgggcact ctcaggatga
     5281 tgtcctctcc gtcccgccaa gccagtttca cgttgtaggc ggacaggctc agcacagcgt
     5341 cgtgctcctg ggtcaggtgg ccaggcagct ggtgggctct cttggcgttg tcgataaagt
     5401 gcaggatctc ggtccggcta gaaggattca ggtagccggg gatgcttgtc agttggccca
     5461 ggtacttcac ttctttctcg atgtaatcag acagcagtct gtcgggttcc actctctcgg
     5521 gcaggctcag gaccacggtg tgcagaggtc ttctttctgt aactttttca tgggccttct
     5581 tgtcccggct cttctcgccc ttcaggaaca cccgcttaaa aggagacacg atgccaggtt
     5641 tcttgccctt cttgccctct tcttccatgg atccgagctc ggtacccttg tacagctcgt
     5701 ccatgccgtg agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct
     5761 tctcgttggg gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca
     5821 gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc
     5881 cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg
     5941 ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc
     6001 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
     6061 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
     6121 ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
     6181 agcggctgaa gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg
     6241 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     6301 cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
     6361 tgggcaccac cccggtgaac agctcctcgc ccttgctcat ggtggcaagc ttaagtttaa
     6421 acgctagcca gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc
     6481 agtgggttct ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct
     6541 accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt
     6601 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
     6661 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
     6721 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
     6781 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
     6841 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
     6901 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
     6961 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
     7021 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa
     7081 tcaatgtcaa cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct
     7141 aagcagattc ttcatgcaat tgtcggtcaa gccttgcctt gttgtagctt aaattttgct
     7201 cgcgcactac tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc
     7261 ggcatcagag cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg
     7321 tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     7381 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttca
//
