LOCUS       sequence-242329-        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_deltaC55G-hutRNApro_BsmBIplch-sgRNA_14n...
FEATURES             Location/Qualifiers
     source          1..3559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    63..111
                     /note="synthetic polyadenylation signal"
     misc_feature    125..216
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     165..184
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     misc_RNA        726..801
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    887..1008
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(898..917)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     952..971
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(1285..1873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1365..1384)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2044..2835,2836..2904))
                     /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     2667..2686
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2905..3009)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3036..3491
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3123..3142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3333..3354
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc
       61 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
      121 tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
      181 tccccagtgc aagtgcaggt gccagaacat ttctctatcg atagctagct gaaatattaa
      241 tgtcgcgact attaatattt tcaggctagc catgcttcgc tacacgcgac tattaatatt
      301 ttcaggctag ccatgcttcg ctacacgcga ctattaatat tttcaagcta gccatgcttc
      361 gctacacgcg actattaata ttttcaggct agccatgctt cgctaacgcg actattaata
      421 ttttcaggct agtcatgctt cgctacacgc gactattaat attttcaggc tagccatgct
      481 tcgctacacg cgactattaa tattttcagg ctagccatgc ttcgctacac gcgactatta
      541 atattttcag gctagccatg cttcgctaca cgcgactatt aatattttca ggctagcggg
      601 gggctataaa agggggtggg ggcgttcgtc ctgctatcta gcgtcgcgtt gaccatgcca
      661 acggctcgtt gggaggtccc gggttgaaat cccggacgag cccggagacg ggaattcacg
      721 tctccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
      781 aaagtggcac cgagtcggtg caaccagttt gtgtcggctc gttgggaggt cccgggttga
      841 aatcccggac gagcccggga cggccggccg cttcgagcag acatgataag atacattgat
      901 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
      961 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1021 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1081 aacctctaca aatgtggtaa aatcgataag gatcgagtat gtcgaccgat gcccttgaga
     1141 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     1201 atgatgcaac tcgtaggaca ggtgcatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1261 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1321 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1381 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1441 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1501 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1561 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1621 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1681 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     1741 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1801 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1861 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1921 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     1981 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2041 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2101 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2161 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2221 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2281 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2341 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2401 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2461 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2521 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2581 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2641 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2701 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     2761 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     2821 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     2881 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     2941 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3001 ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag
     3061 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     3121 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3181 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3241 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3301 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3361 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     3421 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3481 gcttacaatt tgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     3541 ggcctcttcg ctattacgc
//
