LOCUS       sequence-429720-        8893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA BFP + Puromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8893)
  TITLE     Plasmids for dCas9-Krab repression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8893)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pLG1 library vector (pU6-sgRNA
            Ef1alpha-Puro-T...
FEATURES             Location/Qualifiers
     source          1..8893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2822..2855)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2859..2879
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        3132..3343
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3344..4282
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4239..4259
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4303..4899
                     /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(4303..4322)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4799..4819
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4909..4962
                     /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             4975..5673
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5693..5726)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5782..6370
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5835..5855)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6253..6264)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6373..6389)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6374..6390)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6899..7079
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7141..7729)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7221..7240)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7900..8691,8692..8760))
                     /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     8523..8542
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8761..8865)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgccctgcag gtctagagat ccgacgcgcc
     2641 atctctaggc ccgcgccggc cccctcgcac ggacttgtgg gagaagctcg gctactcccc
     2701 tgccccggtt aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa
     2761 aagacagata atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc
     2821 tataacttcg tatagcatac attatacgaa gttataaaca gcacaaaagg aaactcaccc
     2881 taactgtaaa gtaattgtgt gttttgagac tataagtatc ccttggagaa ccaccttgtt
     2941 ggaccaggat gggcaccacc cgtttaagag ctaagctgga aacagcatag caagtttaaa
     3001 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctcgag
     3061 tactaggatc cattaggcgg ccgccctgca gggtggataa ccgtattacc gccatgcatg
     3121 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     3181 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     3241 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     3301 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
     3361 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
     3421 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
     3481 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
     3541 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
     3601 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
     3661 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     3721 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     3781 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     3841 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     3901 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     3961 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     4021 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     4081 gcgcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     4141 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     4201 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     4261 aagttttttt cttccatttc aggtgtcgtg agctagccca ccatgaccga gtacaagccc
     4321 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg
     4381 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc
     4441 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4501 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4561 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4621 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4681 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     4741 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     4801 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     4861 ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatcgggaga gggcagagga
     4921 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caccggtcgc caccagcgag
     4981 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac
     5041 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     5101 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc
     5161 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc
     5221 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     5281 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     5341 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc
     5401 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag
     5461 ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc
     5521 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag
     5581 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac
     5641 ctccctagca aactggggca caagcttaat taagaattcg tcgagggacc taataacttc
     5701 gtatagcata cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa
     5761 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     5821 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5881 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     5941 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     6001 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     6061 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     6121 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     6181 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     6241 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     6301 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     6361 gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac
     6421 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     6481 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     6541 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     6601 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     6661 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     6721 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     6781 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     6841 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     6901 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     6961 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     7021 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     7081 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7141 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7201 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7261 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7321 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7381 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7441 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7501 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7561 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7621 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7681 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7741 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7801 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7861 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7921 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7981 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     8041 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     8101 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     8161 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8221 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     8281 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     8341 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     8401 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8461 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8521 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8581 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8641 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8701 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8761 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8821 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8881 agtgccacct gac
//
