LOCUS       sequence-416569-        7190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-TLNRD1 with the K192E and R233E mutations in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7190)
  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 7190)
  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: TLNRD1_2E_eGFP
FEATURES             Location/Qualifiers
     source          1..7190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(154..173)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        345..724
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        725..928
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     879..899
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     973..992
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        973..991
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1033..1746
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(1033..1035,1036..1746)
                     /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(1336..1355)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1683..1704
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2891..2908)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2897..3121
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3167..3595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3254..3273)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3464..3485
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3604..3624)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3609..3938
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3789..3924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3851..3870
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4005..4799
                     /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(4059..4078)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4669..4688
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4973..5094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5010..5029)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5064..5083
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5143..5159)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5143..5159)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5156..5178)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5167..5183
                     /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(5191..5197,5198..5215,5216..5221))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5236..5257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5374..5391)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5545..6133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5625..5644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6304..7095,7096..7164))
                     /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     6927..6946
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7165..7190,1..79))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
       61 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
      121 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      181 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
      241 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      301 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      361 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      421 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      481 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      541 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      601 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      661 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      721 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      781 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      841 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      901 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      961 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
     1021 aagcttgcca ccatgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1081 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1141 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1201 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1261 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1321 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1381 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1441 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1501 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1561 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1621 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1681 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctcacggcat ggacgagctg
     1741 tacaagggta ccgagctcgg atccatggcc tctggcagcg ccggaaaacc taccggcgag
     1801 gccgcatccc ccgcccctgc tagcgctatc ggcggcgcca gcagccagcc cagaaagaga
     1861 ctggtgtccg tgtgcgacca ctgcaagggc aaaatgcagc tggtggccga cctgctgctc
     1921 ctgtccagcg aggctagacc tgtgctgttc gagggcccag cttctagcgg cgccggcgct
     1981 gaaagcttcg agcagtgcag agatacaatc atcgcccgga ccaagggcct gtctatcctg
     2041 acccacgacg tgcagtctca actgaacatg ggcagattcg gcgaagccgg cgacagcctg
     2101 gtggagctgg gcgacctggt tgtgagcctg accgagtgca gcgcccacgc cgcctacctg
     2161 gccgccgtgg ccacacctgg cgcccagcct gcccagcctg gactggtgga ccggtacaga
     2221 gtgaccagat gccggcacga ggtggaacag ggctgcgccg tcctgagggc cacccctctg
     2281 gccgatatga ccccccagct gctcctggaa gtgagccagg gactgagccg caacctggaa
     2341 ttcctgaccg acgcctgcgc cctggcctct gataagagcc gggatcggtt cagcagagag
     2401 cagtttaagc tgggcgtgaa gtgcatgtca acaagcgcca gcgccctgct ggcttgcgtc
     2461 gaggaagtga aggtggcccc tagcgagctg gccagaagcc ggtgcgctct ttttagcggc
     2521 cccctggtcc aggccgtgtc cgccctggtg ggcttcgcca ccgagcctca gttcctggga
     2581 agagccgccg ctgtttctgc cgagggtaag gccgtgcaaa cagccatcct gggcggcgct
     2641 atgagcgtgg tgagcgcctg tgtgctgctg acacagtgtc tgagagatct ggcccagcac
     2701 cccgacggcg gagcaaagat gagcgaccat agagagcggc tgcggaactc tgcctgtgcc
     2761 gtgtctgaag gatgtaccct gctgagccaa gctctgagag aaagatcctc cccaagaaca
     2821 ctgcctccag tgaacagcaa cagcgtgaat taactcgagt ctagagggcc cgtttaaacc
     2881 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     2941 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3001 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3061 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3121 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     3181 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     3241 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     3301 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     3361 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     3421 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     3481 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     3541 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     3601 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     3661 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     3721 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     3781 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3841 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3901 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3961 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4021 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4081 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4141 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4201 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4261 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4321 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4381 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4441 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4501 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4561 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4621 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4681 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4741 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4801 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4861 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4921 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4981 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     5041 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     5101 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     5161 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5221 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5281 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5341 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5401 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5461 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5521 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5581 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5641 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5701 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5761 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5821 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5881 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5941 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6001 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6061 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6121 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6181 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6241 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6301 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6361 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6421 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6481 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6541 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6601 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6661 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6721 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6781 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6841 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6901 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6961 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7021 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7081 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7141 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
//
