LOCUS       sequence-217997-        7670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of a single sgRNA and the fluorophore 
            violet-excited GFP in immune cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7670)
  AUTHORS   LaFleur MW, Nguyen TH, Coxe MA, Yates KB, Trombley JD, Weiss SA, 
            Brown FD, Gillis JE, Coxe DJ, Doench JG, Haining WN, Sharpe AH
  TITLE     A CRISPR-Cas9 delivery system for in vivo screening of genes in the 
            immune system.
  JOURNAL   Nat Commun. 2019 Apr 10;10(1):1668. doi: 10.1038/s41467-019-09656-2.
  PUBMED    30971695
REFERENCE   2  (bases 1 to 7670)
  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 
            Commun."; date: "2019-04-10"; volume: "10(1):1668. doi"; pages: " 
            10.1038/s41467-019-09656-2"
COMMENT     Sequence Label: pXPR_053
FEATURES             Location/Qualifiers
     source          1..7670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..20
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        65..140
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    193..310
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        359..869
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     788..806
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      877..886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(883..885,886..888,889..1602)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLFIQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(928..949)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1189..1208)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1536..1557
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1609..2197
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1662..1682)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2080..2091)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2269..2502
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2580..2701
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2617..2636)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2671..2690
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2741..2876
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2896..2915)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2897..2915)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2925..2942)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2925..2941)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2934..2956)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3083..3538
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3170..3189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3380..3401
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3564..3668
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3669..3737,3738..4529)
                     /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(3887..3906)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4700..5288
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5189..5208
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5442..5459
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5576..5597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5612..5617,5618..5635,5636..5642)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5650..5666
                     /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     5655..5677
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5674..5690
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5674..5690
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5709..5729
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5711..5729
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5757..5983
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5984..6164
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6211..6336
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6829..7062
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7247..7291
                     /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"
     primer_bind     7469..7489
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7640..7659
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 aattgtgagc gctcacaatt atatatcttg tggaaaggac gaaacaccgg agacggacgt
       61 ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      121 aagtggcacc gagtcggtgc ttttttatgc ggaattctcg acctcgagac aaatggcagt
      181 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
      241 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
      301 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg
      361 ggcccgggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg
      421 ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg
      481 ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg
      541 ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg
      601 aagccgcaca tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc
      661 gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg
      721 gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc
      781 ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg
      841 ttgaccgaat caccgacctc tctccccagg ggatccgcca ccatggtgag caagggcgag
      901 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      961 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     1021 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcagc
     1081 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     1141 tccgccatgc ccgaaggcta cgtccaggag cgcaccatca gcttcaagga cgacggcaac
     1201 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1261 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1321 aacagccaca acgtctatat cacggccgac aagcagaaga acggcatcaa ggcgaacttc
     1381 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1441 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgtt catccagtcc
     1501 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1561 gccgccggga tcactcacgg catggacgag ctgtacaagt aacgtacgaa tcaacctctg
     1621 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     1681 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     1741 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     1801 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     1861 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     1921 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     1981 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     2041 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     2101 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtctttg ccttcgccct
     2161 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt
     2221 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     2281 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     2341 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     2401 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     2461 gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat
     2521 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     2581 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2641 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2701 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     2761 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2821 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2881 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     2941 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     3001 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     3061 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3121 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3181 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3241 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3301 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3361 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3421 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3481 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     3541 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3601 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3661 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3721 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3781 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3841 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3901 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3961 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4021 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4081 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4141 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4201 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4261 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4321 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4381 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4441 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4501 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4561 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4621 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4681 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4741 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4801 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4861 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4921 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4981 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5041 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5101 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5161 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5221 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5281 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5341 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5401 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5461 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5521 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5581 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     5641 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     5701 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
     5761 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
     5821 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
     5881 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
     5941 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
     6001 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6061 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6121 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     6181 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     6241 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     6301 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     6361 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     6421 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     6481 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     6541 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     6601 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     6661 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     6721 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     6781 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     6841 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     6901 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     6961 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     7021 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     7081 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     7141 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     7201 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     7261 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     7321 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     7381 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     7441 ccacctccca accccgaggg gacccagaga gggcctattt cccatgattc cttcatattt
     7501 gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa
     7561 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     7621 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
//
