LOCUS       sequence-431904-        8644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for Sp gRNA, contains Sp gRNA scaffold (with 
            truncations) with mU6 promoter. Includes mClover for bacterial 
            screening and GFP for mammalian screening..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8644)
  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 8644)
  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: p103_gRNA_Sp_mClover
FEATURES             Location/Qualifiers
     source          1..8644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1783..1981
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             1999..2179
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2226..2351
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2848..3081
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3266..3310
                     /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    3608..3725
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(3978..4011)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4015..4035
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     RBS             4249..4260
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4267..4269,4270..4272,4273..4983)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTIYFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQNNSIK
                     ANFTIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     terminator      4995..5066
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5082..5109
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     enhancer        5328..5631
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5632..5835
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             join(5869..5871,5872..5874,5875..6588)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6175..6194)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    complement(6608..6641)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6697..7285
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6750..6770)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7168..7179)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7288..7304)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7289..7305)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7814..7994
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(8056..8644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8136..8155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     2041 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     2101 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     2161 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     2221 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     2281 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     2341 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     2401 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     2461 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     2521 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     2581 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     2641 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct
     2701 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     2761 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     2821 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     2881 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     2941 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     3001 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     3061 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     3121 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     3181 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     3241 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     3301 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     3361 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     3421 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     3481 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     3541 tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca
     3601 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     3661 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     3721 attttcgggt ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct
     3781 agagatccga cgcgccatct ctaggcccgc gccggccccc tcgcacggac ttgtgggaga
     3841 agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt aactatagag
     3901 gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta cattttacat
     3961 gataggcttg gatttctata acttcgtata gcatacatta tacgaagtta taaacagcac
     4021 aaaaggaaac tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata agtatccctt
     4081 ggagaaccac cttggagacg gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt
     4141 gtaaatctta tttaataatg tgtgcggcaa ttcacattta atttatgaat gttttcttaa
     4201 catcgcggca actcaagaaa cggcaggttc ggatcttagc tactagagaa agaggagaaa
     4261 tactagatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     4321 ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg gcgagggcga gggcgatgcc
     4381 accatcggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     4441 cccaccctcg tgaccacctt cggctacggc gtggcctgct tcagccgcta ccccgaccac
     4501 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     4561 atctacttca aggacgacgg tacctacaag acccgcgccg aggtgaagtt cgagggcgac
     4621 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     4681 gggcacaagc tggagtacaa cttcaacagc cactacgtct atatcacggc cgacaagcag
     4741 aacaacagca tcaaggctaa cttcaccatc cgccacaacg ttgaggacgg cagcgtgcag
     4801 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     4861 aaccactacc tgagccatca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     4921 atggtcctgc tggagttcgt gaccgccgcc gggattacac atggcatgga cgagctgtac
     4981 aagtaaccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     5041 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc
     5101 ctttctgcgt ttatacgtct ccagagctat gctggaaaca gcatagcaag tttaaataag
     5161 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctcgagtact
     5221 aggatccatt aggcggccgc gtggataacc gtattaccgc catgcattag ttattaatag
     5281 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     5341 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     5401 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     5461 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     5521 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     5581 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     5641 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     5701 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     5761 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     5821 tatataagca gagctggttt agtgaaccgt cagatccgct agcccaccat ggtgagcaag
     5881 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     5941 ggacacaagt tcagcgtgtc tggcgagggc gagggcgatg ccacctacgg caagctgacc
     6001 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     6061 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     6121 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     6181 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     6241 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     6301 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcg
     6361 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     6421 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     6481 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     6541 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaga attcgtcgag
     6601 ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta agggttccgg
     6661 ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     6721 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     6781 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     6841 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     6901 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     6961 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     7021 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     7081 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     7141 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     7201 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     7261 atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag acctagaaaa
     7321 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
     7381 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
     7441 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg
     7501 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg
     7561 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt
     7621 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg
     7681 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga
     7741 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca
     7801 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     7861 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     7921 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     7981 gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     8041 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     8101 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     8161 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     8221 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     8281 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     8341 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     8401 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     8461 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     8521 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     8581 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     8641 tcaa
//
