LOCUS       sequence-451546-        4588 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Donor template for mStayGold insertion into the C-terminus of the 
            TOMM20 locus. For mitochondria visualization. To be co-transfected 
            with sgRNA plasmid px330-PITCh-TOMM20 (Addgene #207789).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4588)
  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 4588)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-Solo-mStayGold-TOMM20
FEATURES             Location/Qualifiers
     source          1..4588
                     /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             1038..1715
                     /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(2367..2383)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2367..2383)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2380..2402)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2391..2407
                     /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(2415..2421,2422..2439,2440..2445))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2460..2481
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2598..2615)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2769..3357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2849..2868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3528..4319,4320..4388))
                     /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     4151..4170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4389..4493)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4561..4579
                     /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 ctggttttag ttacataatt
      421 gactgatgta atattatgta atgatggctg tacacagtgt ctcatgccct ataatcctag
      481 cactcatttg agctcaggag ttcaaggcca gcctgggcaa catggtaaaa ccctgtctcc
      541 accaaaaata aaaaaaaaat tagccgggca tggtggcatg agtctgtggt tccagctact
      601 caggaggctg aggtggggga ggatcgcttg agcccaggag tcagaggttg cagtgagctg
      661 agattgtgct actgcactcc agcctgggtt acagagaccc catctgaatt aaaaacatat
      721 ataatgtaat gatctgcctc ctttgttaac ttgacttttg aaatgggatt gtcagtagta
      781 tgatcattgt tttcttggat gccgactgtg tgtaaagtgt tacattttga attaaatgtc
      841 agaatgggtg aactttacta agattcaatt ctttgaatac aaagagcatt ttattttgaa
      901 gttagaatcc taattaaatg cttatgacac tttaaaaaat tatttttttt ttctttcaga
      961 gaattgtaag tgctcagagc ttagctgaag atgatgttga aggaggaagc ggaggatctg
     1021 gcggtaccgg cggaagcatg gtgtctacag gcgaggaact gtttaccggc gtggtgccct
     1081 tcaagttcca gctgaagggc accatcaacg gcaagagctt caccgtggaa ggcgagggcg
     1141 agggcaatag ccacgagggc agccacaaag gcaagtatgt gtgcaccagc ggcaaactgc
     1201 caatgtcttg ggccgccctg ggaactagct tcggctatgg catgaaatac tataccaagt
     1261 accccagcgg cctgaaaaac tggttccacg aggtgatgcc tgagggcttc acctacgaca
     1321 gacacatcca gtacaagggc gacggcagca tccacgccaa gcaccagcac ttcatgaaga
     1381 acggcaccta ccacaacatc gtggagttca ccggccagga cttcaaggag aacagccccg
     1441 tgctgaccgg cgacatggac gtgagcctgc ccaacgaggt gcagcacatc cccattgatg
     1501 acggcgtgga gtgcacagtg accctgcagt accctctgct gagcgacgaa agcaagtgcg
     1561 tggaagccta tcagaacacc atcatcaagc ccctgcacaa tcagccagcc cccgatgtgc
     1621 catttcactg gatcagaaag cagtacaccc agagcaagga cgacaccgag gagagagacc
     1681 acatcatcca gagcgagacc ctggaggccc acctgggagg ttccctcgag accggtaaga
     1741 gcaagtgatg agaaacaaat gtcaacataa taaaatctca gttaaaaata ttttaaaaat
     1801 tcttggtagt tgagcagctc tgggggaata agggcaaata tgcttgttat gaactacact
     1861 gaaatctacc aaagttaatg tttactttgt gtagatccat ttgtctattt tatttatttt
     1921 tcccagtgaa aagtgtattt tgatagagaa cttttcattc tataaataca ctatgagtta
     1981 ctaaaatatc atggattttg tttattcctg aaacatagtt acatagttaa actgtacata
     2041 tgacatggct tatgttaaaa atacccagtg ctcagttttg aaagataggc aaaaaaaaaa
     2101 aagtatagga gaaactgaag aatgtacact tttttagagg gcacattttg ctgtaaatct
     2161 ggaaatttga tagacttgac tgtgtttgtg aaaactgagc attaaaggtt ttgattgatc
     2221 ctttctttcc atttaatctc tgagacgtaa atatgtgagg tgtgctgctg tgctgggtta
     2281 acagcttcct tccctttctg tgtagcagtc ttgaaatgtt ctgtttaaat cagtaggctt
     2341 aatgtgttaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     2401 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     2461 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2521 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2581 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2641 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2701 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2761 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2821 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2881 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2941 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3001 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3061 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3121 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3181 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3241 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3301 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3361 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3421 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3481 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3541 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3601 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3661 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3721 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3781 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3841 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3901 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3961 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4021 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4081 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4141 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4201 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4261 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4321 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4381 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4441 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4501 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     4561 aataggcgta tcacgaggcc ctttcgtc
//
