LOCUS       sequence-341908-        5438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid to clone recombination arms to create homologous 
            recombination donor vector for C-terminal gene tagging with mTagBFP2
            and selection with Blasticidin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5438)
  AUTHORS   Perez-Leal O, Nixon-Abell J, Barrero CA, Gordon JC, Oesterling J, 
            Rico MC
  TITLE     Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and
            Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, 
            Mitochondrial Dynamics, and Cell Growth.
  JOURNAL   CRISPR J. 2021 Nov 30. doi: 10.1089/crispr.2021.0041.
  PUBMED    34847745
REFERENCE   2  (bases 1 to 5438)
  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: "CRISPR J. 
            2021 Nov 30. doi: 10.1089/crispr.2021.0041."
COMMENT     Sequence Label: FASTHDR-Cterm-mTagBFP2-Blasticidin
FEATURES             Location/Qualifiers
     source          1..5438
                     /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             732..1037
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     CDS             1813..1830
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1846..1902
                     /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"
     CDS             2817..3122
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3217..3233)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3217..3233)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3230..3252)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3241..3257
                     /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(3265..3271,3272..3289,3290..3295))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3310..3331
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3448..3465)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3619..4207)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3699..3718)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4378..5169,5170..5238))
                     /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     5001..5020
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5239..5343)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5411..5429
                     /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 ccagtgggta ccggcttact aaaagccaga
      421 taacagtatg cgtatttgcg cgctgatttt tgcggtataa gaatatatac tgatatgtat
      481 acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg tattacagtg acagttgaca
      541 gcgacagcta tcagttgctc aaggcatata tgatgtcaat atctccggtc tggtaagcac
      601 aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc tggaaagcgg aaaatcagga
      661 agggatggct gaggtcgccc ggtttattga aatgaacggc tcttttgctg acgagaacag
      721 gggctggtga aatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
      781 ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
      841 ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
      901 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg
      961 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     1021 tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtctgca ggtcgacggt
     1081 acccaaggcg gtggagaatt catggtctca aaaggagaag aactcatcaa agaaaatatg
     1141 cacatgaaac tttatatgga ggggacagtg gataatcacc actttaaatg tacttctgag
     1201 ggggagggga aaccgtatga aggcacccag accatgagga ttaaggttgt cgaaggcggg
     1261 ccccttccct tcgccttcga catacttgcg acatccttct tgtatggttc caaaaccttc
     1321 atcaatcata cgcagggcat cccagatttc ttcaaacaga gtttcccaga gggatttact
     1381 tgggagaggg tcacgacgta tgaagatgga ggcgtcttga ccgctaccca ggacaccagt
     1441 cttcaagatg ggtgtttgat atacaacgta aaaataaggg gagttaattt taccagtaac
     1501 gggccagtaa tgcagaaaaa gaccttggga tgggaggctt ttacggaaac cttgtatcct
     1561 gcggatggag gtttggaggg cagaaatgat atggccctga aactcgtcgg tgggtctcat
     1621 ctgatagcaa atgcaaaaac aacatatcga agtaagaagc cagctaagaa ccttaaaatg
     1681 ccaggagttt attacgtgga ttataggctc gaacggatca aggaggccaa caacgagacc
     1741 tatgttgagc aacatgaggt agcagtcgct aggtattgtg atctgccgag caaacttggg
     1801 cataaactga atcaccatca ccatcaccat gaattcggat ctggagcaac aaacttctca
     1861 ctactcaaac aagcaggtga cgtggaggag aatcccgggc cttctagaat gccactgtct
     1921 caggaagaga gcacactgat agagagagca accgcgacca tcaatagcat acctatcagt
     1981 gaagattaca gtgtggccag cgccgcactt tcttctgatg gccggatttt caccggcgtg
     2041 aacgtctatc acttcacggg gggaccatgt gcagagctgg tggtactcgg gacagccgcg
     2101 gccgctgcag ctggcaacct cacatgcatc gtagccatcg ggaacgagaa tcgcggtatc
     2161 ttgtcaccct gtgggagatg caggcaggtt ctgctcgatt tgcacccggg aattaaagca
     2221 atcgtgaagg actccgacgg ccagccaacc gcagtcggca tcagagaatt gctgcctagc
     2281 ggctatgtgt gggaaggctc tagataaatt cgtcagtagg gttgtaaagg tttttctttt
     2341 cctgagaaaa caaccttttg ttttctcagg ttttgctttt tggcctttcc ctagctttta
     2401 aaaaaaaaaa agcaaaagac gctggtggct ggcactcctg gtttccagga cggggttcaa
     2461 gtccctgcgg tgtctttgct tggatccggc ttactaaaag ccagataaca gtatgcgtat
     2521 ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca
     2581 aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt
     2641 tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga
     2701 agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt
     2761 cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct ggtgaaatgc
     2821 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
     2881 gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     2941 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     3001 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     3061 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     3121 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg acggatcccg ggcccgtcga
     3181 ctgcagaggc ctgcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3241 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3301 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3361 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3421 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3481 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3541 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3601 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3661 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3721 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3781 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3841 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3901 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3961 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4021 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4081 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4141 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4201 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4261 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4321 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4381 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4441 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4501 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4561 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4621 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4681 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4741 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4801 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4861 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4921 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4981 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5041 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5101 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5161 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5221 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5281 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5341 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     5401 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
