LOCUS       sequence-242342-        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"
     misc_feature    269..360
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(301..320)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    374..422
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(553..1008)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(690..711)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     902..921
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1035..1139
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1140..1208,1209..2000)
                     /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(1358..1377)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2171..2759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2660..2679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3036..3157
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3073..3092)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3127..3146
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_RNA        complement(3274..3349)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 gcgaagcatg gctagcctga aaatattaat agtcgcgtgt agcgaagcat gactagcctg
       61 aaaatattaa tagtcgcgtt agcgaagcat ggctagcctg aaaatattaa tagtcgcgtg
      121 tagcgaagca tggctagctt gaaaatatta atagtcgcgt gtagcgaagc atggctagcc
      181 tgaaaatatt aatagtcgcg tgtagcgaag catggctagc ctgaaaatat taatagtcgc
      241 gacattaata tttcagctag ctatcgatag agaaatgttc tggcacctgc acttgcactg
      301 gggacagcct attttgctag tttgttttgt ttcgttttgt tttgatggag agcgtatgtt
      361 agtactatcg attcacacaa aaaaccaaca cacagatgta atgaaaataa agatatttta
      421 ttgcggccgc tccaagtacc tcccgtacct taatattact tacttatcat ggtagcttgg
      481 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      541 atggcgaatg gcaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
      601 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
      661 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
      721 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
      781 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
      841 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
      901 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
      961 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     1021 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1081 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1141 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1201 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1261 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1321 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1381 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1441 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1501 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1561 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1621 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1681 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1741 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1801 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1861 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1921 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1981 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2041 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2101 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2161 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2221 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2281 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2341 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2401 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2461 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2521 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2581 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2641 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2701 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2761 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgc
     2821 acctgtccta cgagttgcat cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     2881 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacatactcg atccttatcg
     2941 attttaccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     3001 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     3061 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3121 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgctcg aagcggccgg
     3181 ccgtcccggg ctcgtccggg atttgaaccc gggacctctc gcaccctaag cgagaatcat
     3241 acccctagac caacgagccg acacaaactg gttgcaccga ctcggtgcca ctttttcaag
     3301 ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaacg gagacgtgaa
     3361 ttcccgtctc cgggctcgtc cgggatttga acccgggacc tctcgcaccc taagcgagaa
     3421 tcatacccct agaccaacga gccgttggca tggtcaacgc gacgctagat agcaggacga
     3481 acgcccccac ccccttttat agccccccgc tagcctgaaa atattaatag tcgcgtgtag
     3541 cgaagcatgg ctagcctgaa aatattaata gtcgcgtgta gcgaagcatg gctagcctga
     3601 aaatattaat agtcgcgtgt a
//
