LOCUS       sequence-242336-        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"
     misc_feature    373..464
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(405..424)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    478..526
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(657..1112)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(794..815)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1006..1025
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1139..1243
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1244..1312,1313..2104)
                     /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     complement(1462..1481)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2275..2863
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2764..2783
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3140..3261
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3177..3196)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3231..3250
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_RNA        complement(3347..3422)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 aaaatattaa tagtcgcgtg tagcgaagca tggctagcct gaaaatatta atagtcgcgt
       61 gtagcgaagc atggctagcc tgaaaatatt aatagtcgcg tgtagcgaag catggctagc
      121 ctgaaaatat taatagtcgc gtgtagcgaa gcatgactag cctgaaaata ttaatagtcg
      181 cgttagcgaa gcatggctag cctgaaaata ttaatagtcg cgtgtagcga agcatggcta
      241 gcttgaaaat attaatagtc gcgtgtagcg aagcatggct agcctgaaaa tattaatagt
      301 cgcgtgtagc gaagcatggc tagcctgaaa atattaatag tcgcgacatt aatatttcag
      361 ctagctatcg atagagaaat gttctggcac ctgcacttgc actggggaca gcctattttg
      421 ctagtttgtt ttgtttcgtt ttgttttgat ggagagcgta tgttagtact atcgattcac
      481 acaaaaaacc aacacacaga tgtaatgaaa ataaagatat tttattgcgg ccgctccaag
      541 tacctcccgt accttaatat tacttactta tcatggtagc ttgggctggc gtaatagcga
      601 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcaaat
      661 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
      721 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
      781 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
      841 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
      901 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
      961 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     1021 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     1081 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     1141 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1201 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1261 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1321 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1381 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1441 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1501 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1561 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1621 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1681 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1741 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1801 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1861 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1921 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     1981 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2041 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2101 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2161 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2221 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2281 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2341 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2401 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     2461 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2521 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2581 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2641 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2701 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2761 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2821 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2881 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgcacctgt cctacgagtt
     2941 gcatcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3001 tgaaggctct caagggcatc ggtcgacata ctcgatcctt atcgatttta ccacatttgt
     3061 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3121 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3181 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3241 caaactcatc aatgtatctt atcatgtctg ctcgaagcgg ccggccgtcc cgggctcgtc
     3301 cgggatttga acccgggacc tcccaacgag ccgacacaaa ctggttgcac cgactcggtg
     3361 ccactttttc aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa
     3421 acggagacgt gaattcccgt ctccgggctc gtccgggatt tcaacccggg acctcccaac
     3481 gagccgttgg catggtcaac gcgacgctag atagcaggac gaacgccccc accccctttt
     3541 atagcccccc gctagcctg
//
