LOCUS       sequence-341877-        5855 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 HaloTag 
            and selection with Puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5855)
  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 5855)
  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-HaloTag-Puromycin
FEATURES             Location/Qualifiers
     source          1..5855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1287..1343)
                     /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             complement(1359..1376)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1377..2261)
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             complement(2277..2297)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             complement(2362..2667)
                     /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(3004..3021)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3004..3020)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3013..3035)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3229..3248)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3348..3370
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3408..3426)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3494..3598
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3599..3667,3668..4459)
                     /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     complement(3817..3836)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4630..5218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5119..5138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5372..5389
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5506..5527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5542..5547,5548..5565,5566..5572)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5580..5596
                     /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)."
     primer_bind     5585..5607
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5604..5620
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5604..5620
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(join(5715..5855,1..165))
                     /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"
ORIGIN
        1 atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag
       61 ggggatcacc atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag
      121 acgataacgg ctctctcttt tataggtgta aaccttaaac tgcatttcac cagcccctgt
      181 tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct
      241 gattttccgc tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc
      301 ttaccagacc ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc
      361 aactgtcact gtaatacgct gcttcatagc atacctcttt ttgacatact tcgggtatac
      421 atatcagtat atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg
      481 gcttttagta agccggatcc aagcaaagac accgcaggga cttgaacccc gtcctggaaa
      541 ccaggagtgc cagccaccag cgtcttttgc tttttttttt ttaaaagcta gggaaaggcc
      601 aaaaagcaaa acctgagaaa acaaaaggtt gttttctcag gaaaagaaaa acctttacaa
      661 ccctactgac gaatttatct agatgccccc ggttttcttg tcatgcacca agtcctcggt
      721 ccttctggaa cttcgacatc ggcggtgacg gtaaatccaa ggcgctcgta aaagggcaga
      781 tttcgtgggg cactagtctc gaggaaagcc gggactcctg ccctctcggc tgcctccacc
      841 cctggcaaga ctactgcgga accgagtccc ttgccctggt ggtccgggct aaccccaact
      901 gtggccaaaa accaagcggg ctccttcggc ctgtggggtg ccaggagtcc ctccatctgc
      961 tgctgggcag ccagtcggct gccagacagc tccgccatcc ggggcccgat ctctgcaaac
     1021 acagccccgg cctcgacgct ctccggcgtg gtccacacgg ccaccgcggc gccatcgtcg
     1081 gcgacccaga ctttgcctat gtccagcccc acccgggtca ggaaaagctc ctgcaattca
     1141 gtcactctct cgatatgccg atctgggtcg accgtatgcc tggttgcagg atagtcggca
     1201 aacgcggctg cgagggtccg cacggctctg ggcacgtcat ccctggttgc caatctcaca
     1261 gtgggtttat actctgtcat tctagaaggc ccgggattct cctccacgtc acctgcttgt
     1321 ttgagtagtg agaagtttgt tgctccagat ccgaattcat ggtgatggtg atggtgaccg
     1381 gaaatctcca gagtagacag ccagcgcgcg atctcgctgc cgatcaggtc cgggttgtct
     1441 tcttgcagca gattcagacc cgggccgatg tccacagcct tgcagttagg caggcttttg
     1501 gccaggcgag cggcttcggc cggtgggatc agaacgcctg gggtgcccca gaacagcagc
     1561 ttcgggacag gggactggtg cagccagtcc atgtattctt cgaccagcgc gacgatgttc
     1621 gctggctcac cggcgattgg cagctcgttt gggaagcgcc acagtggctc gcggtcaaca
     1681 ggattcagga acggctcgcg gtaatggtcc atctcgactt cagtcagcgg gcggacgaca
     1741 cccatcggca gcgtaccctc gataaaaacg ttctgatcga tgatcagctt gcggccgacg
     1801 tcggtggtgc ggaaggcctg gaaggtctcg cgggcaaatt ctggccattc gtcccaggtc
     1861 gggatagggc ggatgaactc cataaatgca atacctttga cgcgctctgg attgcgcttg
     1921 gcccagtgga aacccagagc ggagccccag tcgtgaatga ccaggacgac ctcttccaga
     1981 cccagggctt cgatgaaggc atccatgaag cggacgtggt cgtcgaagaa ataacccagg
     2041 tctggtttgt cggatttgcc cataccgatc aggtctggag caatgcagcg atgggtcggt
     2101 gcaacatgcg ggatgatgtt gcgccacacg taggaggagg tcgggttacc gtgcaggaac
     2161 agcacagggg tgccatcgcg cggaccaaca tcgacgtagt gcatgcgctc gcccaggact
     2221 tccacataat gggggtcgaa tggaaagcca gtaccgattt cggaaccatc gttatcgctc
     2281 tgaaagtaca gatcctcgaa ttctccaccg ccttgggtac cgtcgacctg cagactggct
     2341 gtgtataagg gagcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat
     2401 gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga ccggcacact
     2461 ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca ccgggtaaag
     2521 ttcacgggag actttatctg acagcagacg tgcactggcc agggggatca ccatccgtcg
     2581 cccgggcgtg tcaataatat cactctgtac atccacaaac agacgataac ggctctctct
     2641 tttataggtg taaaccttaa actgcatttc accagcccct gttctcgtca gcaaaagagc
     2701 cgttcatttc aataaaccgg gcgacctcag ccatcccttc ctgattttcc gctttccagc
     2761 gttcggcacg cagacgacgg gcttcattct gcatggttgt gcttaccaga ccggagatat
     2821 tgacatcata tatgccttga gcaactgata gctgtcgctg tcaactgtca ctgtaatacg
     2881 ctgcttcata gcatacctct ttttgacata cttcgggtat acatatcagt atatattctt
     2941 ataccgcaaa aatcagcgcg caaatacgca tactgttatc tggcttttag taagccggta
     3001 cccactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3061 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3121 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     3181 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     3241 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3301 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3361 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     3421 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3541 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3601 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3661 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3721 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3781 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3841 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3901 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3961 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4021 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4081 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4141 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4201 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4261 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     4321 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4381 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4441 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4501 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4561 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4621 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4681 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4741 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     4801 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4861 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4921 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4981 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5041 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5101 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5161 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5221 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5281 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5341 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     5401 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5461 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     5521 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     5581 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
     5641 atgcaggcct ctgcagtcga cgggcccggg atccgtcgac ctgcagactg gctgtgtata
     5701 agggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt
     5761 tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata
     5821 tcggtggtca tcatgcgcca gctttcatcc ccgat
//
