LOCUS       sequence-341228-        8157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral gRNA delivery.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8157)
  AUTHORS   Labuhn M, Adams FF, Ng M, Knoess S, Schambach A, Charpentier EM, 
            Schwarzer A, Mateo JL, Klusmann JH, Heckl D
  TITLE     Refined sgRNA efficacy prediction improves large- and small-scale 
            CRISPR-Cas9 applications.
  JOURNAL   Nucleic Acids Res. 2018 Feb 16;46(3):1375-1385. doi: 
            10.1093/nar/gkx1268.
  PUBMED    29267886
REFERENCE   2  (bases 1 to 8157)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2018-02-16- 16"; volume: "46"; issue: 
            "3"; pages: "1375-1385"
COMMENT     Sequence Label: SGL40C.EFS.E2Crimson
FEATURES             Location/Qualifiers
     source          1..8157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    235..256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(271..276,277..294,295..301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    309..325
                     /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     314..336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     333..349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     333..349
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     368..388
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        370..388
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        433..812
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        869..1060
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(892..912)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1079..1305
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1306..1486
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1533..1658
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2151..2384
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2569..2613
                     /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    2911..3028
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3079..3327
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3079..3099
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3250..3269
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3489..3700
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3720..3729
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3726..4403
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     4124..4143
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    4425..5013
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4478..4498)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4896..4907)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5085..5318
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5396..5517
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5433..5452)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5487..5506
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5557..5692
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5712..5731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5713..5731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5741..5758)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5741..5757)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5750..5772)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5899..6354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5986..6005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6196..6217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6380..6484
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6485..6553,6554..7345)
                     /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(6703..6722)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7516..8104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8005..8024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
       61 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      121 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      181 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      241 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      301 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      361 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cggtaccgtc
      421 gacctgcagg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      481 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      541 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      601 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      661 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      721 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      781 acatctacgt attagtcatc gctattacca tggcgcgcca tgcagggcct gaaataacct
      841 ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt
      901 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
      961 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     1021 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc ttaagaggcg cgccttaatg
     1081 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     1141 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     1201 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     1261 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     1321 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     1381 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     1441 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     1501 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     1561 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     1621 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     1681 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     1741 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     1801 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     1861 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     1921 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     1981 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     2041 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     2101 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     2161 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     2221 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     2281 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     2341 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     2401 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     2461 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     2521 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     2581 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     2641 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     2701 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     2761 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     2821 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     2881 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2941 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     3001 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     3061 ttggttaatt aaggtaccga gggcctattt cccatgattc cttcatattt gcatatacga
     3121 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     3181 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     3241 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     3301 tatatcttgt ggaaaggacg aaacaccgga gacgtatgtc gacaactcgt ctctgtttaa
     3361 gagctatgct ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa
     3421 aagtggcacc gagtcggtgc ttttttgaat tctcctctag aactcgagtg gctccggtgc
     3481 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     3541 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     3601 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     3661 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtgtcgtgac gcgggatccg
     3721 ccaccatgga tagcactgag aacgtcatca agcccttcat gcgcttcaag gtgcacatgg
     3781 agggctccgt gaacggccac gagttcgaga tcgagggcgt gggcgagggc aagccctacg
     3841 agggcaccca gaccgccaag ctgcaagtga ccaagggcgg ccccctgccc ttcgcctggg
     3901 acatcctgtc cccccagttc ttctacggct ccaaggcgta catcaagcac cccgccgaca
     3961 tccccgacta cctcaagcag tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     4021 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcaccctca
     4081 tctaccacgt gaagttcatc ggcgtgaact tcccctccga cggccccgta atgcagaaga
     4141 agactctggg ctgggagccc tccactgagc gcaactaccc ccgcgacggc gtgctgaagg
     4201 gcgagaacca catggcgctg aagctgaagg gcggcggcca ctacctgtgt gagttcaagt
     4261 ccatctacat ggccaagaag cccgtgaagc tgcccggcta ccactacgtg gactacaagc
     4321 tcgacatcac ctcccacaac gaggactaca ccgtggtgga gcagtacgag cgcgccgagg
     4381 cccgccacca cctgttccag tagtgtacaa cgcgttaagt cgacaatcaa cctctggatt
     4441 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     4501 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     4561 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     4621 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     4681 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     4741 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     4801 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     4861 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     4921 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     4981 cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa
     5041 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     5101 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     5161 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5221 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     5281 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta gttcatgtca
     5341 tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt
     5401 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5461 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5521 tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5581 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5641 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagggacg
     5701 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     5761 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     5821 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     5881 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5941 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     6001 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     6061 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     6121 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     6181 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     6241 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     6301 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     6361 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     6421 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     6481 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     6541 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     6601 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     6661 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6721 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6781 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6841 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6901 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6961 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     7021 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7081 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7141 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7201 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     7261 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     7321 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     7381 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     7441 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     7501 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     7561 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     7621 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     7681 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7741 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7801 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7861 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7921 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7981 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     8041 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     8101 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgc
//
