LOCUS       sequence-214336-        7664 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 7664)
  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 7664)
  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..7664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             66..299
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    371..492
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(408..427)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     462..481
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      532..667
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(687..706)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(688..706)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(716..733)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(716..732)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(725..747)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      874..1329
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(961..980)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1171..1192
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1355..1459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1460..1528,1529..2320)
                     /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(1678..1697)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2491..3079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2980..2999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3233..3250
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3367..3388
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3403..3408,3409..3426,3427..3433)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3441..3457
                     /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     3446..3468
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3465..3481
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3465..3481
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3500..3520
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3502..3520
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3548..3774
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3775..3955
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4002..4127
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4620..4853
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5038..5082
                     /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     5260..5280
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5431..5450
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    5462..5481
                     /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        5526..5601
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    5654..5771
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5820..6330
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6249..6267
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      6338..6347
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6344..6346,6347..6349,6350..7063)
                     /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(6389..6410)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6650..6669)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6997..7018
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    7070..7658
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7123..7143)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7541..7552)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
       61 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg
      121 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      181 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      241 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      301 tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga
      361 gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
      421 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
      481 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc
      541 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
      601 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
      661 ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
      721 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
      781 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
      841 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      901 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      961 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1021 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1081 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1141 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1201 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1261 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1321 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1381 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     1441 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     1501 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     1561 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     1621 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     1681 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     1741 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     1801 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1861 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1921 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1981 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     2041 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     2101 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     2161 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     2221 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     2281 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     2341 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     2401 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2461 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2521 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2581 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     2641 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2701 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2761 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2821 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2881 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2941 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3001 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3061 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3121 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3181 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3241 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     3301 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     3361 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc
     3421 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     3481 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca
     3541 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
     3601 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
     3661 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
     3721 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
     3781 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
     3841 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
     3901 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     3961 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     4021 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     4081 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     4141 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     4201 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     4261 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     4321 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     4381 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     4441 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     4501 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     4561 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     4621 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     4681 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     4741 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     4801 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     4861 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     4921 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     4981 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     5041 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     5101 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     5161 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     5221 tcgtttcaga cccacctccc aaccccgagg ggacccagag agggcctatt tcccatgatt
     5281 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
     5341 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     5401 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     5461 taattgtgag cgctcacaat tatatatctt gtggaaagga cgaaacaccg gagacggacg
     5521 tctctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     5581 aaagtggcac cgagtcggtg cttttttatg cggaattctc gacctcgaga caaatggcag
     5641 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     5701 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     5761 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
     5821 gggcccgggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     5881 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     5941 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     6001 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     6061 gaagccgcac atctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     6121 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     6181 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     6241 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     6301 gttgaccgaa tcaccgacct ctctccccag gggatccgcc accatggtga gcaagggcga
     6361 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     6421 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     6481 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcag
     6541 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     6601 gtccgccatg cccgaaggct acgtccagga gcgcaccatc agcttcaagg acgacggcaa
     6661 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     6721 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     6781 caacagccac aacgtctata tcacggccga caagcagaag aacggcatca aggcgaactt
     6841 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     6901 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctgt tcatccagtc
     6961 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     7021 cgccgccggg atcactcacg gcatggacga gctgtacaag taacgtacga atcaacctct
     7081 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     7141 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     7201 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     7261 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     7321 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     7381 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     7441 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     7501 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     7561 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     7621 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgac
//
