LOCUS       sequence-174170-        5421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-mEGFP sequence for N-terminus tagging of 
            human TJP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5421)
  AUTHORS   Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua MA, 
            Nelson A, Hookway C, Ludmann SA, Mueller IA, Yang R, Horwitz R, 
            Rafelski SM, Gunawardane RN
  TITLE     Systematic gene tagging using CRISPR/Cas9 in human stem cells to 
            illuminate cell organization.
  JOURNAL   Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 
            10.1091/mbc.E17-03-0209. Epub 2017 Aug 16.
  PUBMED    28814507
REFERENCE   2  (bases 1 to 5421)
  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"; doi: 
            "10.1091/mbc.E17-03-0209"; journalName: "Mol Biol Cell"; date: 
            "2017-10-15- 15"; volume: "28"; issue: "21"; pages: "2854-2874"
COMMENT     Sequence Label: AICSDP-23:TJP1-mEGFP
FEATURES             Location/Qualifiers
     source          1..5421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1412..1414,1415..1417,1418..2128)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1457..1478)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1718..1737)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2065..2086
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3200..3216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3200..3216)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3213..3235)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3224..3240
                     /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(3248..3254,3255..3272,3273..3278))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3293..3314
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3431..3448)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3602..4190)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3682..3701)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4361..5152,5153..5221))
                     /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     4984..5003
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5222..5326)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5394..5412
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gacgcgtatt gggatagcct
      421 tggcagtcgg cgccggtgaa cgagagcaac gcttctgacc ctgccggagc tcctcggaga
      481 tgaaagccat gacgcgcctt gcagaaaatg cattccgcct tccgtgggaa caacgccgag
      541 gcacgcggtg acagccgtga ccatgctgtt tgcccagtga aggaaacaac tgtcgggtat
      601 cggctctgcc ggcctttcca gccgcactca tgcatggggc tcaccccatg atgtgcgtgg
      661 cttgtcgagg agcaagtgga caagtctctt aaggaaagct ttggtgcaca ggcgctttct
      721 ccttgggggc gaattctgcc agaccttgga taaaaacaaa caggaagact cgcacggcag
      781 cggaaactgt cttccaagtt acttgggtta cccggctttt ccttccgcgc ttggggtcgg
      841 gaccccggcc gctcgtcccg ccccctcccc cgccgcggcc ccgccccctc cccgcctcgc
      901 ctcgcctcgc ctcgtccagc cccgcccccg ccgggccggg catgctcagt gggccgggcc
      961 ggcaggtttg cgtggccgct gagttgccgg cgccggctga gccagcggac gccgcgttcc
     1021 ttggcggccg ccggttcccg ggaagttacg tggcgaagcc ggcttccgag gagacgccgg
     1081 gaggccacgg gtgctgctga cgggcgggcg accgggcgag gccgacgtgg ccgggctgcg
     1141 aaagctgcgg gaggccgagt gggtggccgc gctcggaggg aggtgccggt cgggcgcgcc
     1201 ccgtggagaa gacccgggcg gggcgggcgc ttcccggact tttgtccgag ttgaattccc
     1261 tccccctggg ccgggccctt ccggccgccc ccgcccgtgc cccgctcgct ctcgggagat
     1321 gtttatttgg gctgtggcgt gaggagcggg cgggccagcg ccgcggagtt tcgggtccga
     1381 ggagttgcgc gcggcgctgg agagagacaa gatggtgagc aagggcgagg agctgttcac
     1441 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1501 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1561 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1621 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1681 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1741 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1801 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1861 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1921 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1981 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa
     2041 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2101 cactctcggc atggacgagc tgtacaagag cggcctgaga agcagagccc tggagagaga
     2161 caagagtgcc agagctgcgg ccgcaaaggt gagaacctcc gcggccgcca gggccagacc
     2221 gggccgaccg tcgccgcccg cccaccggca tctggcccgc gtcccgccct ccctcgctgg
     2281 cggctgtctg ggccccgggg cggcggggtg ggcagggctg gcgcggggcc gcgggccgcg
     2341 ggccgcgggc cgcggggagc ccctcgggcg ggggcggcgc gggccgcact gggggcggcc
     2401 ggggaggggg ctgcgggcgc ccggccgccg tactgggcag gtgcatagct gccggcgcct
     2461 gtgcctggct gcggctcgct gagggcgggg acacgcaaca ggtccctcgc ggagaaactc
     2521 ggctccagtg agggttcggg ggctggaagc cggctctcag cgggtcgggg cttggggtgc
     2581 cacctcctgc tggccgggag ctgctgtctt tggaggagtg gttggtcccc ggcgaaaccc
     2641 tgtagtttcg atctgatgtc actccctgcg gtatgcgcac gccagcgata aggctttgag
     2701 actgcaaaac actccactca gcctgtgagg cgtagtaggt cgggttttct ttcatgctgt
     2761 attacttatt taaggtaact ttgaaaataa cctctttaac atttaataat ttaacttgaa
     2821 ttaaactttc acaagtaata caaagtattc ctacgaatgg acaataagat gagcacttaa
     2881 aaattagtaa aggccggtga gttcagccga aaaaagtaac gtttttcctg ttacttttcc
     2941 tatgtgctct gaaatattat tgcattttcc cattgctttg aaactaactt gtgtattaca
     3001 ttaaaaagcc aaagttcctg aaaaacagct aggatgctcc tcccattttg tatattaatt
     3061 ttttcatcat aaaatagtac ttgttatttc aaacaaagga atacagaaat gtgaggagta
     3121 aaaaatctcc cctttaaaga atatcaattc attacttcaa atagtatccc aatggcgcgc
     3181 cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3241 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3301 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3361 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3421 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3481 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3541 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3601 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3661 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3721 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3781 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3841 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3901 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3961 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4021 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4081 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4141 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4201 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4261 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4321 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4381 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4441 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4501 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4561 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4621 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4681 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4741 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4801 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4861 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4921 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4981 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5041 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5101 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5161 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5221 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5281 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5341 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5401 gtatcacgag gccctttcgt c
//
