LOCUS       sequence-431903-        8614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for Sa gRNA, contains Sa gRNA scaffold (with 
            truncations) with mU6 promoter. Includes mClover for bacterial 
            screening and mScarlet for mammalian screening..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8614)
  AUTHORS   Pacalin NM, Steinhart Z, Shi Q, Belk JA, Dorovskyi D, Kraft K, 
            Parker KR, Shy BR, Marson A, Chang HY
  TITLE     Bidirectional epigenetic editing reveals hierarchies in gene 
            regulation.
  JOURNAL   Nat Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3.
  PUBMED    38760566
REFERENCE   2  (bases 1 to 8614)
  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 
            Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3."
COMMENT     Sequence Label: p102_gRNA_Sa_mClover
FEATURES             Location/Qualifiers
     source          1..8614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(78..182)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(257..276)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        448..827
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        829..1027
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             1045..1225
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1272..1397
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1894..2127
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2312..2356
                     /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    2654..2771
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(3024..3057)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     3061..3081
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     RBS             3294..3305
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(3312..3314,3315..3317,3318..4028)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTIYFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQNNSIK
                     ANFTIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3357..3378)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3965..3986
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      4040..4111
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4127..4154
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        4168..4243
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        4368..4671
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4672..4875
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4909..5604
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(5042..5060)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5310..5329
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5624..5657)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5713..6301
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5766..5786)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6184..6195)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6304..6320)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6305..6321)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6830..7010
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7072..7660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7152..7171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7831..8614,1..8,9..77))
                     /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     8454..8473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
       61 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
      121 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
      181 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
      241 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
      301 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
      361 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      421 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      481 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      541 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      601 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      661 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      721 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      781 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      841 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      901 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      961 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1021 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
     1081 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1141 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1201 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1261 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1321 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1381 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1441 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1501 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1561 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1621 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1681 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1741 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1801 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1861 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1921 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1981 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     2041 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     2101 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     2161 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     2221 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     2281 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     2341 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     2401 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     2461 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2521 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2581 atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag
     2641 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2701 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2761 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc
     2821 cgctctagag atccgacgcg ccatctctag gcccgcgccg gccccctcgc acggacttgt
     2881 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact
     2941 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt
     3001 ttacatgata ggcttggatt tctataactt cgtatagcat acattatacg aagttataaa
     3061 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataagta
     3121 tcccttggag aaccaccttg agacggaaag tgaaacgtga tttcatgcgt cattttgaac
     3181 attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta tgaatgtttt
     3241 cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta gagaaagagg
     3301 agaaatacta gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     3361 tcgagctgga cggcgacgta aacggccaca agttcagcgt ccgcggcgag ggcgagggcg
     3421 atgccaccat cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     3481 cctggcccac cctcgtgacc accttcggct acggcgtggc ctgcttcagc cgctaccccg
     3541 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     3601 gcaccatcta cttcaaggac gacggtacct acaagacccg cgccgaggtg aagttcgagg
     3661 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     3721 tcctggggca caagctggag tacaacttca acagccacta cgtctatatc acggccgaca
     3781 agcagaacaa cagcatcaag gctaacttca ccatccgcca caacgttgag gacggcagcg
     3841 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     3901 ccgacaacca ctacctgagc catcagtccg ccctgagcaa agaccccaac gagaagcgcg
     3961 atcacatggt cctgctggag ttcgtgaccg ccgccgggat tacacatggc atggacgagc
     4021 tgtacaagta accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     4081 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     4141 tgggcctttc tgcgtttata cgtctccgtt atagtactct ggaaacagaa tctactataa
     4201 caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg agattttttt ctcgagtact
     4261 aggatccatt aggcggccgc gtggataacc gtattaccgc catgcattag ttattaatag
     4321 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     4381 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     4441 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     4501 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     4561 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     4621 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     4681 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     4741 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     4801 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     4861 tatataagca gagctggttt agtgaaccgt cagatccgct agcccaccat ggtgagcaag
     4921 ggcgaggcag tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac
     4981 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     5041 gccaagctga aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct
     5101 cagttcatgt acggctccag ggccttcacc aagcaccccg ccgacatccc cgactactat
     5161 aagcagtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     5221 gccgtgaccg tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag
     5281 ctccgcggca ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg
     5341 gaagcgtcca ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg
     5401 gccctgcgcc tgaaggacgg cggccgctac ctggcggact tcaagaccac ctacaaggcc
     5461 aagaagcccg tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc
     5521 cacaacgagg actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc
     5581 ggcggcatgg acgagctgta caaggaattc gtcgagggac ctaataactt cgtatagcat
     5641 acattatacg aagttataca tgtttaaggg ttccggttcc actaggtaca attcgatatc
     5701 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     5761 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     5821 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     5881 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     5941 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     6001 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     6061 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     6121 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     6181 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     6241 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     6301 catcgatacc gtcgacctcg atcgagacct agaaaaacat ggagcaatca caagtagcaa
     6361 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg
     6421 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
     6481 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     6541 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta
     6601 cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc
     6661 agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc
     6721 tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag
     6781 ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg
     6841 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     6901 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     6961 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gcatgtgagc
     7021 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     7081 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     7141 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     7201 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     7261 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     7321 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     7381 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     7441 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     7501 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7561 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7621 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     7681 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     7741 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     7801 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     7861 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     7921 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     7981 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     8041 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     8101 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     8161 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     8221 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     8281 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     8341 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     8401 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     8461 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     8521 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     8581 ctgatcttca gcatctttta ctttcaccag cgtt
//
