LOCUS       sequence-170765-        7755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral expression vector for wtGFP S65T.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7755)
  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 7755)
  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: pGH311_wtGFP S65T
FEATURES             Location/Qualifiers
     source          1..7755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..6,7..720)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(46..67)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(307..326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     654..675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    810..1398
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(863..883)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1281..1292)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1401..1417)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1402..1418)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             1927..2107
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(2169..2757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2249..2268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2928..3719,3720..3788))
                     /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     3551..3570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3789..3893)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3968..3987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4159..4538
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4540..4738
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4693..4713
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4756..4936
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4983..5108
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5605..5838
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6023..6067
                     /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    6365..6482
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6585..6796
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          6797..7735
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     7692..7712
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
ORIGIN
        1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
       61 ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac
      121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      181 ctcgtgacca ccttcaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      421 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
      481 ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
      541 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
      601 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
      661 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
      721 tctagagttg aaccgactag taagccaggt cgagacatgt ttaagggttc cggttccact
      781 aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
      841 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
      901 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
      961 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     1021 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     1081 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     1141 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     1201 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     1261 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     1321 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     1381 tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga aaaacatgga
     1441 gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa
     1501 gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac
     1561 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     1621 cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc
     1681 cctgattggc agaactacac accagggcca gggatcagat atccactgac ctttggatgg
     1741 tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac
     1801 acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta
     1861 gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac
     1921 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     1981 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     2041 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     2101 tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2161 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2221 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2281 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2341 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2401 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2461 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2521 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2581 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2641 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2701 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2761 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2821 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2881 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2941 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3001 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3061 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3901 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     3961 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     4021 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     4081 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     4141 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     4201 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     4261 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     4321 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     4381 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     4441 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     4501 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     4561 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     4621 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     4681 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg
     4741 cgttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     4801 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     4861 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     4921 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
     4981 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
     5041 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
     5101 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
     5161 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     5221 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     5281 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     5341 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     5401 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat
     5461 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     5521 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     5581 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     5641 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     5701 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     5761 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     5821 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     5881 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     5941 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     6001 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     6061 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     6121 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     6181 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     6241 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     6301 attagtgaac ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc
     6361 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     6421 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     6481 ttcgggttta ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctagg
     6541 gccgcgtgga taaccgtatt accgccatgc atgtgcccgt cagtgggcag agcgcacatc
     6601 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     6661 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     6721 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     6781 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     6841 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     6901 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     6961 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     7021 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     7081 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     7141 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     7201 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     7261 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     7321 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     7381 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     7441 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     7501 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     7561 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     7621 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     7681 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     7741 gtgagctagc ccacc
//
