LOCUS       sequence-413745-        3775 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Empty N-terminus donor cassette. Integrate homology arms to target 
            the N-terminus insertion of a Blast-2A-miRFP670nano3 cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3775)
  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 3775)
  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-Blast-miRFP670nano3
FEATURES             Location/Qualifiers
     source          1..3775
                     /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..860
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             870..926
                     /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(933..966)
                     /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             990..1031
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1554..1570)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1554..1570)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1567..1589)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1578..1594
                     /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(1602..1608,1609..1626,1627..1632))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1647..1668
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1785..1802)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1956..2544)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2036..2055)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2715..3506,3507..3575))
                     /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     3338..3357
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3576..3680)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3748..3766
                     /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 aagcctttgt
      481 ctcaagaaga atccaccctc attgaaagag caacggctac aatcaacagc atccccatct
      541 ctgaagacta cagcgtcgcc agcgcagctc tctctagcga cggccgcatc ttcactggtg
      601 tcaatgtata tcattttact gggggacctt gtgcagaact cgtggtgctg ggcactgctg
      661 ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat cggaaatgag aacaggggca
      721 tcttgagccc ctgcggacgg tgccgacagg tgcttctcga tctgcatcct gggatcaaag
      781 ccatagtgaa ggacagtgat ggacagccga cggcagttgg gattcgtgaa ttgctgccct
      841 ctggttatgt gtgggagggc ggatccggag ctactaactt cagcctgctg aagcaggccg
      901 gagacgtgga ggagaaccct ggaccttcta gaataacttc gtatagcata cattatacga
      961 acggtagcac gcgtggaggt ggcggttccg gcaagcccat ccccaacccc ctgctgggcc
     1021 tggacagcac cggaggtggc ggatcaatgg caaacctgga caagatgctg aacaccaccg
     1081 tgaccgaggt gcgcaagttc ctgcaagcag acagagtgtg cgtgttcaag ttcgaggaag
     1141 attactccgg caccgtcagc cacgaagccg tggacgacag atggattagc atcctgaaaa
     1201 cccaggtgca ggacagatac ttcatggaaa ccagaggcga ggaatacgtc cacggcagat
     1261 accaggccat cgccgacatc tacacagcca atctggtcga gtgctacaga gacctgctga
     1321 tcgagtttca ggtgcgggcc attctggctg tccccatcct gcaaggcaag aagctgtggg
     1381 gcctgctggt ggcccaccaa ctggccggcc ctcgggagtg gcagacctgg gaaatcgact
     1441 tcctgaaaca gcaagccgtg gtgatgggca tcgccatcca gcagagcggt ggaggcggat
     1501 ctctcgaggg aggaagcgga ggatctggcg gtaccaagct tggcgtaatc atggtcatag
     1561 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1621 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1681 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1741 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1801 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1861 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1921 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1981 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2041 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2101 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2161 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2221 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2281 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2341 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2401 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2461 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2521 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2581 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2641 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2701 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2761 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2821 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     2881 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     2941 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3001 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3061 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3121 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3181 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3241 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3301 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3361 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3421 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3481 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3541 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3601 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3661 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     3721 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
