LOCUS       sequence-413761-        4006 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Empty N-terminus donor cassette. Integrate homology arms to target 
            the N-terminus insertion of a Zeo-2A-mScarlet cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4006)
  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 4006)
  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-Zeo-mScarlet
FEATURES             Location/Qualifiers
     source          1..4006
                     /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..836
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     CDS             846..902
                     /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(909..942)
                     /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             966..1007
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1023..1718
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1156..1174)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1424..1443
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1785..1801)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1785..1801)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1798..1820)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1809..1825
                     /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(1833..1839,1840..1857,1858..1863))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1878..1899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2016..2033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2187..2775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2267..2286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2946..3737,3738..3806))
                     /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     3569..3588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3807..3911)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3979..3997
                     /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 tagcatggcc aagttgacca
      481 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
      541 ggctcgggtt cagccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
      601 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
      661 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
      721 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
      781 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggacggat
      841 ccggagctac taacttcagc ctgctgaagc aggccggaga cgtggaggag aaccctggac
      901 cttctagaat aacttcgtat agcatacatt atacgaacgg tagcacgcgt ggaggtggcg
      961 gttccggcaa gcccatcccc aaccccctgc tgggcctgga cagcaccgga ggtggcggat
     1021 caatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag gtgcacatgg
     1081 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     1141 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttctcctggg
     1201 acatcctgtc ccctcagttc atgtacggct ccagggcctt caccaagcac cccgccgaca
     1261 tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     1321 tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac ggcaccctga
     1381 tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta atgcagaaga
     1441 agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc gtgctgaagg
     1501 gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg gacttcaaga
     1561 ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc gaccgcaagt
     1621 tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa cgctccgagg
     1681 gccgccactc caccggcggc atggacgagc tgtacaaggg tggaggcgga tctctcgagg
     1741 gaggaagcgg aggatctggc ggtaccaagc ttggcgtaat catggtcata gctgtttcct
     1801 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     1861 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     1921 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     1981 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2041 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2101 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2161 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2221 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2281 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2341 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2401 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2461 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2521 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2581 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2641 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2701 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2761 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2821 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2881 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2941 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3001 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3061 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3121 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3181 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3241 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3301 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3361 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3421 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3481 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3541 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3601 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3661 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3721 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3781 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3841 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3901 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     3961 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
