LOCUS       sequence-242341-        3559 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for tRNA-released sgRNA. BsmBI flanked placeholder 
            for spacer cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3559)
  AUTHORS   Knapp DJHF, Michaels YS, Jamilly M, Ferry QRV, Barbosa H, Milne TA, 
            Fulga TA
  TITLE     Decoupling tRNA promoter and processing activities enables specific 
            Pol-II Cas9 guide RNA expression.
  JOURNAL   Nat Commun. 2019 Apr 2;10(1):1490. doi: 10.1038/s41467-019-09148-3.
  PUBMED    30940799
REFERENCE   2  (bases 1 to 3559)
  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: "2019-04-2"; volume: "10(1):1490. doi"; pages: " 
            10.1038/s41467-019-09148-3"
COMMENT     Sequence Label: 8xCTS1_delta-hutRNApro_BsmBIplch-sgRNA_14nt
            Bu...
FEATURES             Location/Qualifiers
     source          1..3559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..92
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     41..60
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     misc_RNA        602..677
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    763..884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(774..793)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     828..847
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(1161..1749)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1241..1260)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1920..2711,2712..2780))
                     /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     2543..2562
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2781..2885)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2912..3367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2999..3018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3209..3230
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3498..3546
                     /note="synthetic polyadenylation signal"
ORIGIN
        1 aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc
       61 cagtgcaagt gcaggtgcca gaacatttct ctatcgatag ctagctgaaa tattaatgtc
      121 gcgactatta atattttcag gctagccatg cttcgctaca cgcgactatt aatattttca
      181 ggctagccat gcttcgctac acgcgactat taatattttc aagctagcca tgcttcgcta
      241 cacgcgacta ttaatatttt caggctagcc atgcttcgct aacgcgacta ttaatatttt
      301 caggctagtc atgcttcgct acacgcgact attaatattt tcaggctagc catgcttcgc
      361 tacacgcgac tattaatatt ttcaggctag ccatgcttcg ctacacgcga ctattaatat
      421 tttcaggcta gccatgcttc gctacacgcg actattaata ttttcaggct agcggggggc
      481 tataaaaggg ggtgggggcg ttcgtcctgc tatctagcgt cgcgttgacc atgccaacgg
      541 ctcgttggga ggtcccgggt tcaaatcccg gacgagcccg gagacgggaa ttcacgtctc
      601 cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      661 tggcaccgag tcggtgcaac cagtttgtgt cggctcgttg ggaggtcccg ggttcaaatc
      721 ccggacgagc ccgggacggc cggccgcttc gagcagacat gataagatac attgatgagt
      781 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      841 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
      901 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
      961 tctacaaatg tggtaaaatc gataaggatc gagtatgtcg accgatgccc ttgagagcct
     1021 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     1081 tgcaactcgt aggacaggtg catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1141 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1201 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1261 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1321 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1381 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1441 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1501 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1561 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1621 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1681 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1741 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1801 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1861 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1921 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1981 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2041 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2101 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2161 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2221 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2281 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2341 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2401 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2461 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2521 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2581 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2641 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2701 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2761 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2821 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2881 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     2941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3121 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3241 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     3361 acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     3421 tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgggag
     3481 gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt
     3541 tgtgtgaatc gatagtact
//
