LOCUS       sequence-413751-        4252 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Empty N-terminus donor cassette. Integrate homology arms to target 
            the N-terminus insertion of a Puro-2A-moxGFP cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4252)
  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 4252)
  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-N-Puro-moxGFP
FEATURES             Location/Qualifiers
     source          1..4252
                     /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"
     protein_bind    complement(423..456)
                     /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             465..1061
                     /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(465..484)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     961..981
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1071..1127
                     /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"
     protein_bind    complement(1134..1167)
                     /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)."
     CDS             1191..1232
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(1248..1250,1251..1253,1254..1964)
                     /codon_start=1
                     /product="monomeric GFP variant optimized for use in 
                     oxidizing environments such as the secretory pathway 
                     (Costantini et al., 2015)"
                     /label=moxGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFISTTGKLPVPWPTLVTTLTYGVQSFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1293..1314)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1901..1922
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2031..2047)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2031..2047)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2044..2066)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2055..2071
                     /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(2079..2085,2086..2103,2104..2109))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2124..2145
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2262..2279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2433..3021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2513..2532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3192..3983,3984..4052))
                     /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     3815..3834
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4053..4157)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4225..4243
                     /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 cggcaagagc aagaccggta
      421 gctaccgttc gtatagcata cattatacga agttatctgc tagcatgacc gagtacaagc
      481 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
      541 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
      601 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
      661 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
      721 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
      781 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
      841 ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
      901 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
      961 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     1021 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cggatccgga gctactaact
     1081 tcagcctgct gaagcaggcc ggagacgtgg aggagaaccc tggaccttct agaataactt
     1141 cgtatagcat acattatacg aacggtagca cgcgtggagg tggcggttcc ggcaagccca
     1201 tccccaaccc cctgctgggc ctggacagca ccggaggtgg cggatcaatg gtgagcaagg
     1261 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtgaacg
     1321 gccacaagtt ctccgtgcgg ggcgagggcg agggcgatgc caccaacggc aagctgaccc
     1381 tgaagttcat cagcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1441 tgacctacgg cgtgcagagc ttctcccgct accccgacca catgaagcgc cacgacttct
     1501 tcaagagcgc catgcccgaa ggctacgtcc aggagcgcac catctccttc aaggacgacg
     1561 gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1621 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1681 acttcaactc ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca
     1741 acttcaagat ccgccataac gtggaggacg gctccgtgca gctcgccgac cactaccagc
     1801 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgtccaccc
     1861 agtccaagct gtccaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1921 tgaccgccgc cgggatcact cacggcatgg acgagctgta caagggtgga ggcggatctc
     1981 tcgagggagg aagcggagga tctggcggta ccaagcttgg cgtaatcatg gtcatagctg
     2041 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2101 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2161 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2221 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2281 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2341 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2401 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2461 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2521 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2581 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2641 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2701 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2761 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2821 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2881 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2941 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3001 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3061 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3121 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3181 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3241 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3301 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3361 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3421 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3481 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3541 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3601 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3661 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3721 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3781 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3841 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3901 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3961 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4021 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4081 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4141 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     4201 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
