LOCUS       sequence-375123-        7878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7878)
  TITLE     Cantley Lab plasmids
REFERENCE   2  (bases 1 to 7878)
  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"
COMMENT     Sequence Label: ipUSEPR-sg-Hs-ACE2-A2
FEATURES             Location/Qualifiers
     source          1..7878
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(21..40)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        212..591
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        593..791
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     746..766
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             809..989
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1036..1161
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1658..1891
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2076..2120
                     /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    2418..2535
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2628..2876
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2628..2648
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2799..2818
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3044..3255
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3306..3902
                     /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(3306..3325)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3802..3822
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3918..3974
                     /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             3975..4688
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     misc_feature    4741..5329
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4794..4814)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5212..5223)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5332..5348)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5333..5349)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5858..6038
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6100..6688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6180..6199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6859..7650,7651..7719))
                     /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     7482..7501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7720..7824)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
       61 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
      121 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      181 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      241 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      301 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      361 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      421 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      481 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      541 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      601 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      661 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      721 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      781 tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg
      841 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      901 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      961 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
     1021 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
     1081 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
     1141 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
     1201 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     1261 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1321 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1381 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1441 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1501 gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1561 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     1621 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     1681 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     1741 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     1801 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     1861 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     1921 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     1981 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     2041 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     2101 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     2161 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     2221 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     2281 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
     2341 acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg
     2401 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2461 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2521 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gttagtaccg
     2581 ggcccgggtc gactagtgat cagtgtgagg gagtgtaaag ctggtttgag ggcctatttc
     2641 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     2701 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2761 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2821 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcaa
     2881 aggcgagaga tagttggttt aagagctatg ctggaaacag catagcaagt ttaaataagg
     2941 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttaa accgctgttc
     3001 ctaggaatcc cgaggccttc gagtggctcc ggtgcccgtc agtgggcaga gcgcacatcg
     3061 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg
     3121 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     3181 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     3241 gccgccagaa cacaggtgtc gtgacgcggg acccgacatt agcgctacag cttaagcggg
     3301 ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg
     3361 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc
     3421 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     3481 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc
     3541 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     3601 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     3661 aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag ggcaagggtc
     3721 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     3781 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     3841 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     3901 ccgctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc gacgtggagg
     3961 agaaccccgg ccccatggtg tctaagggcg aagagctgat taaggagaac atgcacatga
     4021 agctgtatat ggagggcacc gtgaacaacc accacttcaa gtgcacatcc gagggcgaag
     4081 gcaagcccta cgagggcacc cagaccatga gaatcaaggt ggtcgagggc ggccctctcc
     4141 ccttcgcctt cgacatcctg gctaccagct tcatgtacgg cagcagaacc ttcatcaacc
     4201 acacccaggg catccccgac ttctttaagc agtccttccc tgagggcttc acatgggaga
     4261 gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc agcctccagg
     4321 acggctgcct catctacaac gtcaagatca gaggggtgaa cttcccatcc aacggccctg
     4381 tgatgcagaa gaaaacactc ggctgggagg ccaacaccga gatgctgtac cccgctgacg
     4441 gcggcctgga aggcagaagc gacatggccc tgaagctcgt gggcgggggc cacctgatct
     4501 gcaacttcaa gaccacatac agatccaaga aacccgctaa gaacctcaag atgcccggcg
     4561 tctactatgt ggaccacaga ctggaaagaa tcaaggaggc cgacaaagag acctacgtcg
     4621 agcagcacga ggtggctgtg gccagatact gcgacctccc tagcaaactg gggcacaaac
     4681 ttaattgaaa acgttgttgt ttggggttga attactcttc tagaccgcgg cgcgcccgat
     4741 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     4801 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4861 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4921 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4981 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     5041 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     5101 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     5161 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     5221 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     5281 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
     5341 cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac
     5401 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac
     5461 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     5521 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     5581 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
     5641 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
     5701 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
     5761 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
     5821 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
     5881 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5941 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     6001 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca
     6061 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6121 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6181 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6241 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6301 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6361 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6421 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6481 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6541 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6601 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6661 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6721 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6781 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6841 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6901 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6961 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     7021 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7081 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7141 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7201 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7261 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7321 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7381 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7441 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7501 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7561 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7621 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7681 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7741 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7801 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     7861 atcgggagat ctcccgat
//
