LOCUS       sequence-354429-        7479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retrovirus for delivery of one sgRNA (empty, bbsI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7479)
  AUTHORS   Henriksson J, Chen X, Gomes T, Ullah U, Meyer KB, Miragaia R, Duddy 
            G, Pramanik J, Yusa K, Lahesmaa R, Teichmann SA
  TITLE     Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive 
            Crosstalk between Activation and Differentiation.
  JOURNAL   Cell. 2019 Jan 7. pii: S0092-8674(18)31569-1. doi: 
            10.1016/j.cell.2018.11.044.
  PUBMED    30639098
REFERENCE   2  (bases 1 to 7479)
  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: "Cell. 2019 
            Jan 7. pii: S0092-8674(18)31569-1. doi: 10.1016/j.cell.2018.11.044."
COMMENT     Sequence Label: pMSCV-U6sgRNA(BbsI)-PGKpuro2ABFP
FEATURES             Location/Qualifiers
     source          1..7479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     75..94
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             182..778
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(182..201)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     678..698
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             797..850
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             851..1552
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     LTR             1638..2152
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    2314..2330
                     /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)."
     promoter        complement(join(2338..2344,2345..2362,2363..2368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2383..2404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2521..2538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2692..3280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2772..2791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3451..4242,4243..4311))
                     /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     4074..4093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4312..4416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4484..4502
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4540..4562)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4662..4681
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4875..4897
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5025..5044
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             5244..5760
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    5824..6165
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             6232..6648
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6578..6600
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6616..6632
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        6759..7007
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6759..6779
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6930..6949
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7024..7099
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        join(7122..7479,1..142)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(7157..7180)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc
       61 ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc
      121 ttcctcatct ccgggccttt cgacctgcat ccatctagat ctcgagcagc tgaagcttac
      181 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
      241 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
      301 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
      361 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
      421 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
      481 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
      541 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
      601 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
      661 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
      721 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg
      781 cggcgggtcc ggaggagagg gcagaggaag tctcctaaca tgcggtgacg tggaggagaa
      841 tcctggccca atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
      901 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
      961 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     1021 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     1081 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     1141 ggacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     1201 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     1261 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     1321 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     1381 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     1441 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     1501 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt gagcggccgc
     1561 taggtacctt taagaccaat gacttacaag gatcgataaa ataaaagatt ttatttagtc
     1621 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa
     1681 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt
     1741 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     1801 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta
     1861 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     1921 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     1981 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg
     2041 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     2101 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca
     2161 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga
     2221 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg
     2281 acagcgcaga aagagctggg gagaattgtg aaattgttat ccgctcacaa ttccacacaa
     2341 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2401 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2461 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2521 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2581 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2641 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2701 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2761 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2821 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2881 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2941 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3001 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3061 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3121 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3181 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3241 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3301 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3361 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3421 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3481 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3541 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3601 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3661 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3721 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3781 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3841 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3901 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3961 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4021 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4081 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4141 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4201 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4261 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4321 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4381 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4441 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4501 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     4561 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4621 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     4681 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4741 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     4801 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     4861 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc
     4921 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     4981 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5041 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5101 aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact
     5161 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta
     5221 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     5281 taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat
     5341 caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag
     5401 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca
     5461 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc
     5521 cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg
     5581 agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt
     5641 cgcccgggta cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc
     5701 gctgatcctt gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt
     5761 tggaggttcc accgagattt ggagacccct gcccagggac caccgacccc cccgccggga
     5821 ggtaagctgg ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc
     5881 atctaatgtt tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc
     5941 cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac
     6001 tttgggggcc gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg
     6061 tcaggatatg tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa
     6121 tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc
     6181 gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact
     6241 gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac
     6301 aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca
     6361 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt
     6421 aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg
     6481 acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag
     6541 cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc
     6601 ccgcctcgat cctcccttta tccagccctc actccttctc taggcgccgg aattcagatc
     6661 ttacgtagct agcggatccc aattgcacag ggacagcaga gatccagttt ggttagtacc
     6721 gggccctacg cgttactcga gccaaggtcg ggcaggaaga gggcctattt cccatgattc
     6781 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     6841 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     6901 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     6961 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa
     7021 gacgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     7081 agtggcaccg agtcggtgct ttttttggat ccaattctac cgggtagggg aggcgctttt
     7141 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     7201 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     7261 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     7321 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     7381 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     7441 gctttgctcc ttcgctttct gggctcagag gctgggaag
//
