LOCUS       sequence-414795-        5206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for mNeon insertion into the N-terminus of the ACTB 
            locus for actin visualization. To be co-transfected with sgRNA 
            plasmid px330-PITCh-ACTB Addgene #207748.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5206)
  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 5206)
  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-mNeon-ACTB
FEATURES             Location/Qualifiers
     source          1..5206
                     /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             1416..2123
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1671..1680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2985..3001)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2985..3001)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2998..3020)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3009..3025
                     /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(3033..3039,3040..3057,3058..3063))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3078..3099
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3216..3233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3387..3975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3467..3486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4146..4937,4938..5006))
                     /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     4769..4788
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5007..5111)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5179..5197
                     /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 ccctataaaa cccagcggcg
      421 cgacgcgcca ccaccgccga gaccgcgtcc gccccgcgag cacagagcct cgcctttgcc
      481 gatccgccgc ccgtccacac ccgccgccag gtaagcccgg ccagccgacc ggggcaggcg
      541 gctcacggcc cggccgcagg cggccgcggc cccttcgccc gtgcagagcc gccgtctggg
      601 ccgcagcggg gggcgcatgg ggggggaacc ggaccgccgt ggggggcgcg ggagaagccc
      661 ctgggcctcc ggagatgggg gacaccccac gccagttcgg aggcgcgagg ccgcgctcgg
      721 gaggcgcgct ccgggggtgc cgctctcggg gcgggggcaa ccggcggggt ctttgtctga
      781 gccgggctct tgccaatggg gatcgcaggg tgggcgcggc ggagcccccg ccaggcccgg
      841 tgggggctgg ggcgccattg cgcgtgcgcg ctggtccttt gggcgctaac tgcgtgcgcg
      901 ctgggaattg gcgctaattg cgcgtgcgcg ctgggactca aggcgctaac tgcgcgtgcg
      961 ttctggggcc cggggtgccg cggcctgggc tggggcgaag gcgggctcgg ccggaagggg
     1021 tggggtcgcc gcggctcccg ggcgcttgcg cgcacttcct gcccgagccg ctggccgccc
     1081 gagggtgtgg ccgctgcgtg cgcgcgcgcc gacccggcgc tgtttgaacc gggcggaggc
     1141 ggggctggcg cccggttggg agggggttgg ggcctggctt cctgccgcgc gccgcgggga
     1201 cgcctccgac cagtgtttgc cttttatggt aataacgcgg ccggcccggc ttcctttgtc
     1261 cccaatctgg gcgcgcgccg gcgccccctg gcggcctaag gactcggcgc gccggaagtg
     1321 gccagggcgg gggcgacctc ggctcacagc gcgcccggct attctcgcaa ctgacaatgg
     1381 gcaagagcaa gaccggtagc ggaggtggcg gttccatggt gagcaagggc gaggaggata
     1441 acatggcctc tctcccagcg acacatgagt tacacatctt tggctccatc aacggtgtgg
     1501 actttgacat ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc
     1561 tgaagtccac caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt
     1621 atggcttcca tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg
     1681 tagatggctc cggataccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta
     1741 ctgttaacta ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg
     1801 ggactggttt ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt
     1861 gcaggtcgaa gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt
     1921 acaccactgg aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca
     1981 agccaatggc ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc
     2041 tcaagcactc caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga
     2101 tgggcatgga cgagctgtac aagggtggag gcggatctgg aggaagcgga ggatctggcg
     2161 gtaccgatga tgatatcgca gcgctcgtcg tcgacaacgg ctccggcatg tgcaaggccg
     2221 gcttcgcggg cgacgatgcc ccccgggccg tcttcccctc catcgtgggg cgccccaggc
     2281 accaggtagg ggagctggct gggtggggca gccccgggag cgggcgggag gcaagggcgc
     2341 tttctctgca caggagcctc ccggtttccg gggtgggggc tgcgcccgtg ctcagggctt
     2401 cttgtccttt ccttcccagg gcgtgatggt gggcatgggt cagaaggatt cctatgtggg
     2461 cgacgaggcc cagagcaaga gaggcatcct caccctgaag taccccatcg agcacggcat
     2521 cgtcaccaac tgggacgaca tggagaaaat ctggcaccac accttctaca atgagctgcg
     2581 tgtggctccc gaggagcacc ccgtgctgct gaccgaggcc cccctgaacc ccaaggccaa
     2641 ccgcgagaag atgacccagg tgagtggccc gctacctctt ctggtggccg cctccctcct
     2701 tcctggcctc ccggagctgc gccctttctc actggttctc tcttctgccg ttttccgtag
     2761 gactctcttc tctgacctga gtctcctttg gaactctgca ggttctattt gctttttccc
     2821 agatgagctc tttttctggt gtttgtctct ctgactaggt gtctaagaca gtgttgtggg
     2881 tgtaggtact aacactggct cgtgtgacaa ggccatgagg ctggtgtaaa gcggccttgg
     2941 agtgtgtatt aagtaggcgc acagtaaagc ttggcgtaat catggtcata gctgtttcct
     3001 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3061 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3121 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3181 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3241 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3301 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3361 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3421 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3481 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3541 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3601 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3661 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3721 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3781 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3841 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3901 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3961 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4021 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4081 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4141 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4201 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4261 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4321 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4381 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4441 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4501 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4561 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4621 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4681 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4741 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4801 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4861 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4921 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4981 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5041 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5101 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     5161 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
