LOCUS       sequence-416568-        7190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-TLNRD1 with the L191T and A225T 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_2T_eGFP
FEATURES             Location/Qualifiers
     source          1..7190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(159..747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(239..258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(918..1709,1710..1778))
                     /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     1541..1560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1779..1883)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1958..1977)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2149..2528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2529..2732
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2683..2703
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2777..2796
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2777..2795
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2837..3550
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(2837..2839,2840..3550)
                     /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(2879..2900)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3140..3159)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3487..3508
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4695..4712)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4701..4925
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4971..5399
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5058..5077)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5268..5289
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5408..5428)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5413..5742
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5593..5728
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5655..5674
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5809..6603
                     /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(5863..5882)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6473..6492
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6777..6898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6814..6833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6868..6887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6947..6963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6947..6963)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6960..6982)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6971..6987
                     /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(6995..7001,7002..7019,7020..7025))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7040..7061
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(7178..7190,1..5))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
       61 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
      121 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      181 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      241 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
      301 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
      361 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
      421 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      481 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
      541 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
      601 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
      661 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
      721 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
      781 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
      841 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
      901 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
      961 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1021 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1081 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1141 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1201 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1261 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     1321 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     1381 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     1441 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     1501 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     1561 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     1621 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     1681 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     1741 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     1801 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     1861 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
     1921 tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat
     1981 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     2041 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     2101 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     2161 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     2221 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     2281 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     2341 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     2401 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     2461 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     2521 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     2581 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     2641 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     2701 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
     2761 actggcttat cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa
     2821 acttaagctt gccaccatga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2881 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2941 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     3001 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     3061 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     3121 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     3181 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     3241 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     3301 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     3361 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     3421 gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg
     3481 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactcacg gcatggacga
     3541 gctgtacaag ggtaccgagc tcggatccat ggccagtggc agcgccggca agcccaccgg
     3601 cgaggccgct agccccgccc ctgcttctgc tatcggcggc gcttctagcc agcccagaaa
     3661 gcggctggtg tccgtgtgcg accactgtaa aggcaaaatg cagctggtgg ccgacctgct
     3721 gctgctgtct agcgaggcca gacctgtgct gttcgagggc cccgctagca gcggcgccgg
     3781 agctgagagc ttcgagcagt gcagagatac aatcatcgcc agaaccaagg gcctgagcat
     3841 cctgacacac gacgtgcaga gccaactgaa catgggcaga ttcggagaag ccggcgatag
     3901 cctggtggaa ctgggcgacc tggtcgtgtc cctgaccgag tgcagcgccc acgccgccta
     3961 cctggccgcc gttgctaccc caggcgccca gcctgcccag cctggcctgg tcgacagata
     4021 cagagtgacc agatgcagac acgaggtgga acagggctgc gccgtgctgc gggccacccc
     4081 actggctgat atgacacctc agctgctgct ggaagtgtct cagggactgt ccagaaacac
     4141 caagttcctg accgacgcct gcgccctggc ctctgataag tcccgggacc ggttcagcag
     4201 ggaacagttc aagctgggcg tgaaatgcat gtctacaagc accagcgccc tgctcgcttg
     4261 cgtgcgggaa gtgaaggtgg cccctagcga gctggcccgg agcagatgcg ccctttttag
     4321 cggccctctg gtgcaagccg tgtccgcatt agttggattt gccacagagc ctcagttcct
     4381 gggcagagcc gccgctgtct ccgccgaggg caaggccgtg cagaccgcca tcctgggtgg
     4441 cgctatgagc gtggtgagcg cctgtgtgct gctgacccag tgtctgagag atctggccca
     4501 acaccccgac ggcggcgcca agatgagcga ccatagagag cggctgcgca actcagcctg
     4561 cgccgtgtct gagggatgta ccctgctcag ccaggccctg cgggaaagaa gcagccctag
     4621 aacactgcct cctgtgaata gcaactctgt gaactaactc gagtctagag ggcccgttta
     4681 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4741 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4801 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4861 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     4921 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     4981 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5041 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5101 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5161 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5221 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5281 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5341 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     5401 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     5461 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     5521 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     5581 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     5641 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     5701 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     5761 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     5821 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     5881 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     5941 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     6001 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     6061 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     6121 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     6181 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     6241 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     6301 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     6361 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     6421 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     6481 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     6541 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     6601 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     6661 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     6721 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     6781 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6841 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6901 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     6961 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     7021 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     7081 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     7141 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
//
