LOCUS       sequence-164935-        7766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral expression vector for wtGFP S65T-Q80H.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7766)
  AUTHORS   Hess GT, Fresard L, Han K, Lee CH, Li A, Cimprich KA, Montgomery SB,
            Bassik MC
  TITLE     Directed evolution using dCas9-targeted somatic hypermutation in 
            mammalian cells.
  JOURNAL   Nat Methods. 2016 Oct 31. doi: 10.1038/nmeth.4038.
  PUBMED    27798611
REFERENCE   2  (bases 1 to 7766)
  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: "Nat 
            Methods. 2016 Oct 31. doi: 10.1038/nmeth.4038."
COMMENT     Sequence Label: pGH314_wtGFP S65T, Q80H
FEATURES             Location/Qualifiers
     source          1..7766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     781..801
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             844..1024
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1695..1928
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2113..2157
                     /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    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2675..2886
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          2887..3825
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3782..3802
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             join(3846..3848,3849..3851,3852..4565)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKHHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3891..3912)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4152..4171)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4499..4520
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    4655..5243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4708..4728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5126..5137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5246..5262)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5247..5263)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5772..5952
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6014..6602)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6094..6113)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6773..7564,7565..7633))
                     /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     7396..7415
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7634..7738)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctagg gccgcgtgga
     2641 taaccgtatt accgccatgc atgtgcccgt cagtgggcag agcgcacatc gcccacagtc
     2701 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     2761 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     2821 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     2881 acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct
     2941 tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg
     3001 gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct
     3061 tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc
     3121 gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg
     3181 acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt
     3241 tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg
     3301 aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg
     3361 cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg
     3421 gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg
     3481 agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg
     3541 aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg
     3601 tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag
     3661 gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct
     3721 tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt
     3781 ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtgagctagc
     3841 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     3901 tggacggcga cgtaaacggc cacaagttca gcgtgtctgg cgagggcgag ggcgatgcca
     3961 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     4021 ccaccctcgt gaccaccttc acctacggcg tgcagtgctt cagccgctac cccgaccaca
     4081 tgaagcacca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     4141 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     4201 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     4261 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     4321 agaacggcat caaggcgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     4381 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     4441 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     4501 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     4561 agtaatctag agttgaaccg actagtaagc caggtcgaga catgtttaag ggttccggtt
     4621 ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     4681 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     4741 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     4801 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     4861 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     4921 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     4981 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     5041 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg
     5101 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     5161 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     5221 ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac
     5281 atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag
     5341 cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga
     5401 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     5461 taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct
     5521 acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg
     5581 gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag
     5641 agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag
     5701 tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc
     5761 cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     5821 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     5881 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     5941 aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     6001 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     6061 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     6121 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     6181 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6241 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6301 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6361 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6421 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6481 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6541 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6601 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6661 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6721 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6781 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6841 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6901 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6961 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7021 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7081 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7141 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     7201 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7261 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7321 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7381 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7441 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7501 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7561 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7621 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7681 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7741 catttccccg aaaagtgcca cctgac
//
