LOCUS       sequence-345023-        6570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses K164A/H165A CoV2 nsp1 in mammalian cells. This contains a 
            3XFLAG Halo tag fusion to nsp1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6570)
  AUTHORS   Mendez AS, Ly M, Gonzalez-Sanchez AM, Hartenian E, Ingolia NT, Cate 
            JH, Glaunsinger BA
  TITLE     The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in 
            suppression of cellular gene expression and preservation of viral 
            gene expression.
  JOURNAL   Cell Rep. 2021 Oct 19;37(3):109841. doi: 
            10.1016/j.celrep.2021.109841. Epub 2021 Sep 30.
  PUBMED    34624207
REFERENCE   2  (bases 1 to 6570)
  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: "Cell Rep.";
            date: "2021-10-19"; volume: "37(3):109841. doi"; pages: " 
            10.1016/j.celrep.2021.109841. Epub 2021 Sep 30"
COMMENT     Sequence Label: pCDNA4TO-3XFLAG Halo K164A/H165A nsp1
FEATURES             Location/Qualifiers
     source          1..6570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             760..825
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             835..1722
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             1735..1755
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(2344..2361)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2350..2574
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2620..3048
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2707..2726)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2917..2938
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3057..3077)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3062..3391
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3242..3377
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3304..3323
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3439..3486
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3505..3879
                     /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"
     polyA_signal    4009..4130
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4046..4065)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4100..4119
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4179..4195)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4179..4195)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4192..4214)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4203..4219
                     /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(4227..4233,4234..4251,4252..4257))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4272..4293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4410..4427)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4581..5169)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4661..4680)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5340..6131,6132..6200))
                     /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     5963..5982
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6201..6305)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6380..6399)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctta ccaatgatgg actacaagga ccacgacggc
      781 gactacaagg accacgacat cgactacaag gacgacgacg acaagggggc ggccgcagaa
      841 atcggtactg gctttccatt cgaccctcat tatgtggaag tcctgggcga gcgcatgcac
      901 tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc
      961 tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct
     1021 ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac
     1081 cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc
     1141 attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc
     1201 aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa
     1261 tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc
     1321 gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa
     1381 gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg
     1441 cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa
     1501 gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca
     1561 ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag
     1621 gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc
     1681 agcgagatcg cgcgctggct gtcgacgctc gagatttccg gcgagccaac cactgaggat
     1741 ctgtactttc agagcgcggc cgagagcctt gtccctggtt tcaacgagaa aacacacgtc
     1801 caactcagtt tgcctgtttt acaggttcgc gacgtgctcg tacgtggctt tggagactcc
     1861 gtggaggagg tcttatcaga ggcacgtcaa catcttaaag atggcacttg tggcttagta
     1921 gaagttgaaa aaggcgtttt gcctcaactt gaacagccct atgtgttcat caaacgttcg
     1981 gatgctcgaa ctgcacctca tggtcatgtt atggttgagc tggtagcaga actcgaaggc
     2041 attcagtacg gtcgtagtgg tgagacactt ggtgtccttg tccctcatgt gggcgaaata
     2101 ccagtggctt accgcaaggt tcttcttcgt aagaacggta ataaaggagc tggtggccat
     2161 agttacggcg ccgatctaaa gtcatttgac ttaggcgacg agcttggcac tgatccttat
     2221 gaagattttc aagaaaactg gaacactgca gctagcagtg gtgttacccg tgaactcatg
     2281 cgtgagctta acggagggta gggccgctcg agtctagagg gcccgtttaa acccgctgat
     2341 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     2401 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     2461 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     2521 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     2581 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     2641 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2701 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     2761 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     2821 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     2881 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     2941 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3001 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     3061 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3121 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3181 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3241 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3301 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     3361 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     3421 cggatctgat cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa
     3481 tacgacaagg tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc
     3541 gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac
     3601 ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg
     3661 gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac
     3721 gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg
     3781 gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc
     3841 ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat
     3901 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     3961 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4021 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4081 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4141 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4201 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4261 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4321 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4381 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4441 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4501 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4561 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4621 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4681 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4741 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4801 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4861 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4921 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4981 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5041 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5101 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5161 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5221 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5281 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5341 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5401 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5461 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5521 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5581 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5641 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5701 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5761 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5821 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5881 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5941 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6001 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6061 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6121 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6181 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6241 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6301 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     6361 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     6421 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     6481 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     6541 ttcgcgatgt acgggccaga tatacgcgtt
//
