LOCUS       sequence-257264-        7034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses MHV68 M7 promoter driving puromycin resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7034)
  AUTHORS   Hartenian EN, Glaunsinger BA
  TITLE     Xrn1 activity broadly represses RNA polymerase II occupancy at 
            mammalian but not viral promoters during herpesvirus infection
  JOURNAL   bioRxiv 585984
REFERENCE   2  (bases 1 to 7034)
  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: "bioRxiv 
            585984"
COMMENT     Sequence Label: pLKO-M7-puro
FEATURES             Location/Qualifiers
     source          1..7034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             176..220
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    441..558
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             955..1554
                     /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(955..974)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1451..1471
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1572..2160
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1625..1645)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2043..2054)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2232..2465
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2543..2664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2580..2599)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2634..2653
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2704..2839
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2859..2878)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2860..2878)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2888..2905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2888..2904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2897..2919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3046..3501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3133..3152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3343..3364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3527..3631
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3632..3700,3701..4492)
                     /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(3850..3869)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4663..5251
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5152..5171
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5405..5422
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5539..5560
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5575..5580,5581..5598,5599..5605)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5613..5629
                     /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     5618..5640
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5637..5653
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5637..5653
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5672..5692
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5674..5692
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5720..5946
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5947..6127
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6174..6299
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6792..7025
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
ORIGIN
        1 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
       61 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
      121 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
      181 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
      241 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
      301 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
      361 gtttcagacc cacctcccaa ccccgactcg aaagcttggc gtaactagat cttgagacaa
      421 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
      481 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
      541 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
      601 ctcgaggggg cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc
      661 taatggacct tctaggtctt gaaaggagtg ggaatcgatt atggcaaacc aggacactgc
      721 tgacagtgac acatgttcca tacttgaccc ccaatcaccc actcccccac cctccgttcc
      781 cccaataact acactctcgt tgtatgacat ttatgccagc atacttagtc cactcgaccc
      841 aaacagcctg gagtcataaa cggtgccaaa tacaagacat ttctaaatcc ttaagtataa
      901 tgtttatatt taactgaata aaagaagtga cgctataaaa ccctcaacgg taccatgacc
      961 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     1021 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     1081 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     1141 aaggtgtggg tcgcggacga cggcgccgcc gtggcggtct ggaccacgcc ggagagcgtc
     1201 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     1261 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     1321 tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     1381 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     1441 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     1501 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgatgtaca
     1561 agtaagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     1621 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     1681 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     1741 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     1801 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     1861 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     1921 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     1981 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     2041 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     2101 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     2161 gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     2221 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
     2281 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     2341 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     2401 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     2461 tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa
     2521 atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag
     2581 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     2641 gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg
     2701 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2761 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     2821 ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg
     2881 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     2941 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     3001 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     3061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3121 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3241 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3361 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3421 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3481 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3541 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3601 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3661 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3721 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3781 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3841 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3901 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3961 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4021 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4081 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4141 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4201 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4261 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4321 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4381 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4441 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4501 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4561 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4621 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4681 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4741 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4801 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4861 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4921 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4981 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5041 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5101 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5161 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5221 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5281 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5341 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5401 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     5461 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5521 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     5581 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     5641 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
     5701 gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg caacatggta
     5761 acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg
     5821 gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct gacatggatt
     5881 ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct agctcgatac
     5941 ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     6001 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6061 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6121 tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc
     6181 gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga
     6241 gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca
     6301 gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa
     6361 agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag
     6421 ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac
     6481 catcccttca gacaggatca gaagaactta gatcattata taatacagta gcaaccctct
     6541 attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg
     6601 aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga
     6661 ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt
     6721 gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga
     6781 gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc
     6841 gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag
     6901 cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg
     6961 ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag
     7021 ctcctgggga tttg
//
