LOCUS       sequence-416573-        6794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express eGFP-TLNRD1 5 helix bundle (TLNRD1 lacking amino acids 
            142-273) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6794)
  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 6794)
  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_5H_eGFP
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     492..511
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(730..834)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(909..928)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1100..1479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1480..1683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1634..1654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1728..1747
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1728..1746
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1788..2501
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             join(1788..1790,1791..2501)
                     /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(2091..2110)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2438..2459
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3250..3267)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3256..3480
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3526..3954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3613..3632)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3823..3844
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3963..3983)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3968..4297
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4148..4283
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4210..4229
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4364..5158
                     /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(4418..4437)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5028..5047
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5332..5453
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5369..5388)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5423..5442
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5502..5518)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5502..5518)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5515..5537)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5526..5542
                     /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(5550..5556,5557..5574,5575..5580))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5595..5616
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5733..5750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5904..6492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5984..6003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6663..6794,1..660,661..729))
                     /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"
ORIGIN
        1 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
       61 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      121 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      181 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      241 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      301 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      361 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      421 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      481 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      541 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      601 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      661 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      721 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      781 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      841 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
      901 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
      961 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     1021 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     1081 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     1141 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1201 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1261 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1321 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1381 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1441 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1501 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1561 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1621 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     1681 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1741 tatagggaga cccaagctgg ctagcgttta aacttaagct tgccaccatg agcaagggcg
     1801 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1861 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1921 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1981 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     2041 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     2101 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     2161 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     2221 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     2281 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     2341 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2401 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     2461 ccgccgccgg gatcactcac ggcatggacg agctgtacaa gggtaccgag ctcggatcca
     2521 tggctagcgg cagcgccggg aagcccactg gcgaggcggc ttctccggct cctgcgagcg
     2581 ccatcggcgg ggccagctcg cagccgcgga agaggctggt atccgtctgc gaccactgca
     2641 agggcaagat gcagctggtg gctgacctgc tgctgctgtc gagcgaggcg cggcccgtgc
     2701 tcttcgaggg ccccgcctcc tctggtgccg gcgccgagtc cttcgagcag tgccgggaca
     2761 ccatcatcgc gcgcaccaag gggctctcca tcctcaccca cgacgtgcag agccagctca
     2821 acatgggccg cttcggggag gcgggggaca gcctggtgga gctgggcgac ctggtggtgt
     2881 cgctgaccga gtgctcggcc cacgcggcct atctggccgc tgtggccacg ccgggcgccc
     2941 aggcggcagc tgtgagcgcc gagggcaagg cggtgcagac cgccatcctg ggcggcgcca
     3001 tgagcgtggt gtcggcctgc gtgctcctga cccagtgcct cagggatctg gcgcagcacc
     3061 ccgacggggg cgccaagatg tcggaccaca gggagaggct gaggaactcg gcctgcgccg
     3121 tgtctgaagg ctgcaccctg ctatctcagg ctttaaggga gaggtcttcg cccaggactt
     3181 taccgccagt gaattccaat tctgtgaatt agctcgagtc tagagggccc gtttaaaccc
     3241 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3301 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3361 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3421 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3481 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     3541 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3601 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3661 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3721 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3781 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3841 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3901 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     3961 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4021 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4081 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4141 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4201 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     4261 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     4321 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4381 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     4441 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     4501 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     4561 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     4621 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     4681 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4741 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4801 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4861 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     4921 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4981 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5041 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5101 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5161 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     5221 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5281 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     5341 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5401 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5461 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5521 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5581 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5641 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5701 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5761 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5821 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5881 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5941 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6001 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6061 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6121 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6181 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6241 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6301 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6361 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6421 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6481 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6541 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6601 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6661 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6721 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6781 cccagtgctg caat
//
