LOCUS       sequence-451538-        4577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mStayGold insertion into the N-terminus of the 
            TJP1 locus. For tight junction visualization. To be co-transfected 
            with sgRNA plasmid px330-TJP1 (Addgene #227299).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4577)
  AUTHORS   Philip R, Sharma A, Matellan L, Erpf AC, Hsu WH, Tkach JM, Wyatt 
            HDM, Pelletier L
  TITLE     qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous 
            tagging.
  JOURNAL   EMBO J. 2024 Dec 12. doi: 10.1038/s44318-024-00337-5.
  PUBMED    39668248
REFERENCE   2  (bases 1 to 4577)
  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: "EMBO J. 
            2024 Dec 12. doi: 10.1038/s44318-024-00337-5."
COMMENT     Sequence Label: qTAG-N-Solo-mStayGold-TJP1
FEATURES             Location/Qualifiers
     source          1..4577
                     /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             1058..1735
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     primer_bind     complement(2356..2372)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2356..2372)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2369..2391)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2380..2396
                     /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(2404..2410,2411..2428,2429..2434))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2449..2470
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2587..2604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2758..3346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2838..2857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3517..4308,4309..4377))
                     /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     4140..4159
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4378..4482)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4550..4568
                     /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 cgtcttccaa gttacttggg
      421 ttacccggct tttccttccg cgcttggggt cgggaccccg gccgctcgtc ccgccccctc
      481 ccccgccgcg gccccgcccc ctccccgcct cgcctcgcct cgcctcgtcc agccccgccc
      541 ccgccgggcc gggcatgctc agtgggccgg gccggcaggt ttgcgtggcc gctgagttgc
      601 cggcgccggc tgagccagcg gacgccgcgt tccttggcgg ccgccggttc ccgggaagtt
      661 acgtggcgaa gccggcttcc gaggagacgc cgggaggcca cgggtgctgc tgacgggcgg
      721 gcgaccgggc gaggccgacg tggccgggct gcgaaagctg cgggaggccg agtgggtggc
      781 cgcgctcgga gggaggtgcc ggtcgggcgc gccccgtgga gaagacccgg gcggggcggg
      841 cgcttcccgg acttttgtcc gagttgaatt ccctccccct gggccgggcc cttccggccg
      901 cccccgcccg tgccccgctc gctctcggga gatgtttatt tgggctgtgg cgtgaggagc
      961 gggcgggcca gcgccgcgga gtttcgggtc cgaggagcct cgcgcggcgc tggagagaga
     1021 caagatgggc aagagcaaga ccggtagcgg cggaagcatg gtgtctacag gcgaggaact
     1081 gtttaccggc gtggtgccct tcaagttcca gctgaagggc accatcaacg gcaagagctt
     1141 caccgtggaa ggcgagggcg agggcaatag ccacgagggc agccacaaag gcaagtatgt
     1201 gtgcaccagc ggcaaactgc caatgtcttg ggccgccctg ggaactagct tcggctatgg
     1261 catgaaatac tataccaagt accccagcgg cctgaaaaac tggttccacg aggtgatgcc
     1321 tgagggcttc acctacgaca gacacatcca gtacaagggc gacggcagca tccacgccaa
     1381 gcaccagcac ttcatgaaga acggcaccta ccacaacatc gtggagttca ccggccagga
     1441 cttcaaggag aacagccccg tgctgaccgg cgacatggac gtgagcctgc ccaacgaggt
     1501 gcagcacatc cccattgatg acggcgtgga gtgcacagtg accctgcagt accctctgct
     1561 gagcgacgaa agcaagtgcg tggaagccta tcagaacacc atcatcaagc ccctgcacaa
     1621 tcagccagcc cccgatgtgc catttcactg gatcagaaag cagtacaccc agagcaagga
     1681 cgacaccgag gagagagacc acatcatcca gagcgagacc ctggaggccc acctgccttg
     1741 gcatgagcct agcgcaagcg cagtgggagg ttccggagga agcggaggat ctggcggtac
     1801 ctccgccaga gctgcggccg ctaaggtgag cgcctccgcg gccgccaggg ccagaccggg
     1861 ccgaccgtcg ccgcccgccc accggcatct ggcccgcgtc ccgccctccc tcgctggcgg
     1921 ctgtctgggc cccggggcgg cggggtgggc agggctggcg cggggccgcg ggccgcgggg
     1981 agcccctcgg gcgggggcgg cgcgggccgc actgggggcg gccggggagg gggctgcggg
     2041 cgcccggccg ccgtactggg caggtgcata gctgccggcg cctgtgcctg gctgcggctc
     2101 gctgagggcg gggacacgca acaggtccct cgcggagaaa ctcggctcca gtgagggttc
     2161 gggggctgga agccggctct cagcgggtcg gggcttgggg tgccacctcc tgctggccgg
     2221 gagctgctgt ctttggagga gtggttggtc cccggcgaaa ccctgtagtt tcgatctgat
     2281 gtcactccct gcggtatgcg cacgccagcg ataaggcttt gagactgcaa aacactcaag
     2341 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2401 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2461 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2521 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2581 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2641 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2701 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2761 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2821 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2881 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2941 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3001 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3061 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3121 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3181 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3241 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3301 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3361 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3421 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3481 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3541 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3601 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3661 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3721 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3781 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3841 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3901 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3961 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4021 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4081 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4141 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4201 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4261 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4321 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4381 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4441 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4501 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     4561 cacgaggccc tttcgtc
//
