LOCUS       sequence-242319-        3613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for tRNA-released synergistic activation mediator 
            gRNA. BsmBI flanked placeholder for spacer cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3613)
  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 3613)
  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_deltaC55A-hutRNApro_BsmBIplch-SAMsgRNA_...
FEATURES             Location/Qualifiers
     source          1..3613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     193..212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    569..690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(606..625)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     660..679
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_RNA        complement(806..824)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        complement(876..894)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_feature    1415..1506
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(1447..1466)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    1520..1568
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(1699..2154)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1836..1857)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2048..2067
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2181..2285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2286..2354,2355..3146)
                     /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(2504..2523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3317..3613,1..292)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
       61 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
      121 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      181 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      241 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      301 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgcacctgtc
      361 ctacgagttg catcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
      421 tgactgggtt gaaggctctc aagggcatcg gtcgacatac tcgatcctta tcgattttac
      481 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
      541 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
      601 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
      661 tggtttgtcc aaactcatca atgtatctta tcatgtctgc tcgaagcggc cggccgtccc
      721 gggctcgtcc gggatttgaa cccgggacct cccaacgagc cgacacaaac tggttgcacc
      781 gactcggtgc cacttggccc tgcagacatg ggtgatcctc atgttggcca agttgataac
      841 ggactagcct tattttaact tgctaggccc tgcagacatg ggtgatcctc atgttggcct
      901 agctctaaaa cggagacgtc cgtctccggg ctcgtccggg attttaaccc gggacctccc
      961 aacgagccgt tggcatggtc aacgcgacgc tagatagcag gacgaacgcc cccaccccct
     1021 tttatagccc cccgctagcc tgaaaatatt aatagtcgcg tgtagcgaag catggctagc
     1081 ctgaaaatat taatagtcgc gtgtagcgaa gcatggctag cctgaaaata ttaatagtcg
     1141 cgtgtagcga agcatggcta gcctgaaaat attaatagtc gcgtgtagcg aagcatgact
     1201 agcctgaaaa tattaatagt cgcgttagcg aagcatggct agcctgaaaa tattaatagt
     1261 cgcgtgtagc gaagcatggc tagcttgaaa atattaatag tcgcgtgtag cgaagcatgg
     1321 ctagcctgaa aatattaata gtcgcgtgta gcgaagcatg gctagcctga aaatattaat
     1381 agtcgcgaca ttaatatttc agctagctat cgatagagaa atgttctggc acctgcactt
     1441 gcactgggga cagcctattt tgctagtttg ttttgtttcg ttttgttttg atggagagcg
     1501 tatgttagta ctatcgattc acacaaaaaa ccaacacaca gatgtaatga aaataaagat
     1561 attttattgc ggccgctcca agtacctccc gtaccttaat attacttact tatcatggta
     1621 gcttgggctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     1681 gcctgaatgg cgaatggcaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     1741 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     1801 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     1861 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     1921 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     1981 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2041 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     2101 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     2161 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2221 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2281 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2341 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2401 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2461 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2521 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2581 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2641 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2701 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2761 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2821 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2881 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2941 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3001 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3061 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3121 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3181 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3241 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3301 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3361 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3421 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     3481 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3541 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3601 gatagttacc gga
//
