LOCUS       sequence-416574-        7037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-TLNRD1 lacking N and Cterm domains (TLNRD1 amino acids 
            35-345) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7037)
  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 7037)
  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_35-345_eGFP
FEATURES             Location/Qualifiers
     source          1..7037
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..52
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(61..489)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(171..192)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     383..402
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    535..759
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     748..765
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1757..2470)
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(join(1757..2467,2468..2470))
                     /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(1799..1820)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2148..2167
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2511..2530)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2512..2530)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2575..2778)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2604..2624)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2779..3158
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3330..3349
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3424..3528
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3529..3597,3598..4389)
                     /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(3747..3766)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4560..5148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5049..5068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5302..5319
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5436..5457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5472..5477,5478..5495,5496..5502)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5510..5526
                     /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     5515..5537
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5534..5550
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5534..5550
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5599..5720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5610..5629)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5664..5683
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5894..6688)
                     /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(6005..6024)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6615..6634
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6755..7037,1..47))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6769..6904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6823..6842)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagaatt
       61 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
      121 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
      181 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
      241 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
      301 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
      361 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
      421 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
      481 agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca gaagccatag
      541 agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct
      601 gccccacccc accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct
      661 cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg
      721 aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt
      781 aaacgggccc tctagactcg agctacctta aagcctgaga tagcagggtg cagccttcag
      841 acacggcgca ggccgagttc ctcagcctct ccctgtggtc cgacatcttg gcgcccccgt
      901 cggggtgctg cgccagatcc ctgaggcact gggtcaggag cacgcaggcc gacaccacgc
      961 tcatggcgcc gcccaggatg gcggtctgca ccgccttgcc ctcggcgctc acagctgccg
     1021 cgcgacccag gaactgcggc tcggtggcga agcctaccag ggcgctcact gcctgcacca
     1081 ggggcccgct gaagagcgca cagcggctgc gcgccagctc actgggcgcc accttcacct
     1141 cgcgcacgca ggccagcagc gccgacgcgc tggtgctcat gcacttgacg cccagcttga
     1201 actgctcccg cgaaaagcgg tcccgtgact tgtcactggc cagggcgcac gcgtccgtca
     1261 ggaacttgag gttgcgcgac aggccctgcg acacctccag cagcagctgc ggcgtcatgt
     1321 cggccagcgg cgtggcgcgc agcacggcgc aaccctgctc cacctcgtgg cggcatcgcg
     1381 tcacgcggta gcggtccacc aggcccggct gcgcgggctg ggcgcccggc gtggccacag
     1441 cggccagata ggccgcgtgg gccgagcact cggtcagcga caccaccagg tcgcccagct
     1501 ccaccaggct gtcccccgcc tccccgaagc ggcccatgtt gagctggctc tgcacgtcgt
     1561 gggtgaggat ggagagcccc ttggtgcgcg cgatgatggt gtcccggcac tgctcgaagg
     1621 actcggcgcc ggcaccagag gaggcggggc cctcgaagag cacgggccgc gcctcgctcg
     1681 acagcagcag caggtcagcc accagctgca tcttgccctt gcagtggtcg cagacggagg
     1741 atccgagctc ggtacccttg tacagctcgt ccatgccgtg agtgatcccg gcggcggtca
     1801 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     1861 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     1921 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     1981 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     2041 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     2101 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     2161 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     2221 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     2281 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     2341 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     2401 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     2461 ccttgctcat ggtggcaagc ttaagtttaa acgctagcca gcttgggtct ccctatagtg
     2521 agtcgtatta atttcgataa gccagtaagc agtgggttct ctagttagcc agagagctct
     2581 gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt
     2641 gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc
     2701 aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta
     2761 ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt
     2821 aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt
     2881 gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt
     2941 ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg
     3001 aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc
     3061 atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt
     3121 aattgattac tattaataac tagtcaataa tcaatgtcaa cgcgtatatc tggcccgtac
     3181 atcgcgaagc agcgcaaaac gcctaaccct aagcagattc ttcatgcaat tgtcggtcaa
     3241 gccttgcctt gttgtagctt aaattttgct cgcgcactac tcagcgacct ccaacacaca
     3301 agcagggagc agatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     3361 ccatagggga tcgggagatc tcccgatccg tcgacgtcag gtggcacttt tcggggaaat
     3421 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3481 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     3541 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     3601 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     3661 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3721 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     3781 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3841 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3901 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3961 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     4021 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     4081 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     4141 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     4201 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     4261 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     4321 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     4381 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     4441 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4501 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4561 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4621 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4681 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4741 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4801 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4861 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4921 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4981 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5041 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5101 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5161 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5221 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5281 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     5341 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     5401 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     5461 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     5521 taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcta gctagaggtc
     5581 gacggtatac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     5641 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     5701 taagctgcaa taaacaagtt ggggtgggcg aagaactcca gcatgagatc cccgcgctgg
     5761 aggatcatcc agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg
     5821 gcggtggaat cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac
     5881 cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     5941 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
     6001 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     6061 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     6121 cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca
     6181 gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg
     6241 cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
     6301 tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg
     6361 caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt
     6421 cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca
     6481 gccacgatag ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct
     6541 tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
     6601 cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac
     6661 ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag
     6721 atccgaaaat ggatatacaa gctcccggga gctttttgca aaagcctagg cctccaaaaa
     6781 agcctcctca ctacttctgg aatagctcag aggcagaggc ggcctcggcc tctgcataaa
     6841 taaaaaaaat tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg
     6901 ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
     6961 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac taattgagat
     7021 gcatgctttg catactt
//
