LOCUS       sequence-452087-        5310 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Donor template for mStayGold-2A-Puro insertion into the C-terminus 
            of the PXN locus. For focal adhesion visualization. To be 
            co-transfected with sgRNA plasmid px330-PITCh-PXN (Addgene #227315).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5310)
  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 5310)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-C-mStayGold-Puro-PXN
FEATURES             Location/Qualifiers
     source          1..5310
                     /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             994..1671
                     /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"
     CDS             1687..1728
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(1750..1783)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             1795..1851
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1858..2454
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1858..1877)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2354..2374
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    complement(2461..2494)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3089..3105)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3089..3105)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3102..3124)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3113..3129
                     /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(3137..3143,3144..3161,3162..3167))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3182..3203
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3320..3337)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3491..4079)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3571..3590)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4250..5041,5042..5110))
                     /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     4873..4892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5111..5215)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5283..5301
                     /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 cggagaacta aataatccca
      421 gccctgtggg aggccgaggt gggaggattg caggaggcca catttgacca gcatgggcaa
      481 tatagtgaga cccccatctc tacaaaaaaa atttatttaa taaaataaaa atgaaaaatg
      541 agcgtttagg acaacacggc acatgggaaa cgcctagcaa gtaggagtca ctccgagcgt
      601 gccgactatg cccacctcgg ccccatcaca cagggtgcag ctctagcccg aggggcagct
      661 ccctgagccc ctctctccgc ctggcaggaa tgcttcacgc cattcgtgaa cggcagcttc
      721 ttcgagcacg acgggcagcc ctactgtgag gtgcactacc acgagcggcg cggctcgctg
      781 tgttctggct gccagaagcc catcaccggc cgctgcatca ccgccatggc caagaagttc
      841 caccccgagc acttcgtctg tgccttctgc ctcaagcagc tcaacaaggg caccttcaag
      901 gagcagaacg acaagcctta ctgtcagaac tgcttcctca agctcttctg cggaggaagc
      961 ggaggatctg gcggtaccgg aggtggcgga agcatggtgt ctacaggcga ggaactgttt
     1021 accggcgtgg tgcccttcaa gttccagctg aagggcacca tcaacggcaa gagcttcacc
     1081 gtggaaggcg agggcgaggg caatagccac gagggcagcc acaaaggcaa gtatgtgtgc
     1141 accagcggca aactgccaat gtcttgggcc gccctgggaa ctagcttcgg ctatggcatg
     1201 aaatactata ccaagtaccc cagcggcctg aaaaactggt tccacgaggt gatgcctgag
     1261 ggcttcacct acgacagaca catccagtac aagggcgacg gcagcatcca cgccaagcac
     1321 cagcacttca tgaagaacgg cacctaccac aacatcgtgg agttcaccgg ccaggacttc
     1381 aaggagaaca gccccgtgct gaccggcgac atggacgtga gcctgcccaa cgaggtgcag
     1441 cacatcccca ttgatgacgg cgtggagtgc acagtgaccc tgcagtaccc tctgctgagc
     1501 gacgaaagca agtgcgtgga agcctatcag aacaccatca tcaagcccct gcacaatcag
     1561 ccagcccccg atgtgccatt tcactggatc agaaagcagt acacccagag caaggacgac
     1621 accgaggaga gagaccacat catccagagc gagaccctgg aggcccacct gggaggtggc
     1681 ggttccggca agcccatccc caaccccctg ctgggcctgg acagcaccgg aggtggcgga
     1741 tcaacgcgtt accgttcgta tagcatacat tatacgaagt tatctggatc cggagctact
     1801 aacttcagcc tgctgaagca ggccggagac gtggaggaga accctggacc ttctagaatg
     1861 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1921 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1981 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     2041 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc
     2101 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     2161 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     2221 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc
     2281 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     2341 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     2401 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcccccggg
     2461 ataacttcgt atagcataca ttatacgaac ggtagcctcg agaccggtaa gagcaagtga
     2521 taggtgccct gcccctgtct ctgcccccct tccccagcca gcatccccaa ctgcgactgt
     2581 gacctagaga cttcacccgg gggtgaaggg gtaaacccga ctgaaactgg aacccttgtc
     2641 ctccgctggt gcgggatgga cagagggccg tgaggggtcc ccctgcttgt cttcacccct
     2701 gccagagcct ctgggccccc tcctccctcc tgtagctctc cctaggctgc ccactctcca
     2761 tcctccccag gggtagaggc tgggggctcc accccagccc atgtacgtcc ccacgaactg
     2821 gcctggccag caccccacac tggagccatc tcttcctcat atttcagcag tgcagccggg
     2881 gggcagggaa gggcaggcag ggtctgttgg ggtctctttt tatccttatt cctcccccga
     2941 cctaattgtc tttgttctgt gattattggg ggacacccgg ctccctccag acaatgccag
     3001 cataaatcca tccatccaaa ggcagagaac caaaggggcc atggaaggtt ctctgtgctc
     3061 ctcctaccct aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3121 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3181 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3241 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3301 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3361 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3421 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     3481 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     3541 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     3601 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     3661 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     3721 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     3781 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     3841 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     3901 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     3961 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4021 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4081 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4141 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4201 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4261 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4321 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4381 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4441 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     4501 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     4561 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     4621 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     4681 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     4741 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     4801 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     4861 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     4921 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     4981 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5041 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5101 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5161 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5221 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     5281 aaaataggcg tatcacgagg ccctttcgtc
//
