LOCUS       sequence-237654-        3621 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 3621)
  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 3621)
  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_WT-hutRNApro_BsmBIplch-sgRNA_14nt
            Buffe...
FEATURES             Location/Qualifiers
     source          1..3621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(468..1259,1260..1328))
                     /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     1091..1110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1329..1433)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1460..1915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1547..1566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1757..1778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    2046..2094
                     /note="synthetic polyadenylation signal"
     misc_feature    2108..2199
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     2148..2167
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     misc_RNA        2740..2815
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    2932..3053
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2943..2962)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2997..3016
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(join(3330..3621,1..297))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3410..3429)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
       61 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
      121 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
      181 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
      241 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
      301 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
      361 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
      421 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
      481 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
      541 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
      601 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
      661 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
      721 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
      781 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
      841 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
      901 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
      961 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     1021 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     1081 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     1141 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     1201 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     1261 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     1321 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     1381 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     1441 ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     1501 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     1561 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     1621 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     1681 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     1741 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     1801 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     1861 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcccat
     1921 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     1981 cgccagccca agctaccatg ataagtaagt aatattaagg tacgggaggt acttggagcg
     2041 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     2101 tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg
     2161 ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgatagcta gctgaaatat
     2221 taatgtcgcg actattaata ttttcaggct agccatgctt cgctacacgc gactattaat
     2281 attttcaggc tagccatgct tcgctacacg cgactattaa tattttcaag ctagccatgc
     2341 ttcgctacac gcgactatta atattttcag gctagccatg cttcgctaac gcgactatta
     2401 atattttcag gctagtcatg cttcgctaca cgcgactatt aatattttca ggctagccat
     2461 gcttcgctac acgcgactat taatattttc aggctagcca tgcttcgcta cacgcgacta
     2521 ttaatatttt caggctagcc atgcttcgct acacgcgact attaatattt tcaggctagc
     2581 ggggggctat aaaagggggt gggggcgttc gtcctgctat ctagcgtcgc gttgaccatg
     2641 ccaacggctc gttggtctag gggtatgatt ctcgcttagg gtgcgagagg tcccgggttc
     2701 aaatcccgga cgagcccgga gacgggaatt cacgtctccg ttttagagct agaaatagca
     2761 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcaacca
     2821 gtttgtgtcg gctcgttggt ctaggggtat gattctcgct tagggtgcga gaggtcccgg
     2881 gttcaaatcc cggacgagcc cgggacggcc ggccgcttcg agcagacatg ataagataca
     2941 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     3001 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     3061 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     3121 agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcg agtatgtcga ccgatgccct
     3181 tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg
     3241 cacttatgat gcaactcgta ggacaggtgc atgtgagcaa aaggccagca aaaggccagg
     3301 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3361 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3421 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3481 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg
     3541 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3601 cagcccgacc gctgcgcctt a
//
