LOCUS       sequence-345701-        2585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden gate vector for gRNA cloning. Contains BbsI sites for 
            insertion of the gRNA spacer as annealed oligos..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2585)
  AUTHORS   Villegas Kcam MC, Tsong AJ, Chappell J
  TITLE     Rational engineering of a modular bacterial CRISPR-Cas activation 
            platform with expanded target range.
  JOURNAL   Nucleic Acids Res. 2021 May 7;49(8):4793-4802. doi: 
            10.1093/nar/gkab211.
  PUBMED    33823546
REFERENCE   2  (bases 1 to 2585)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2021-05-7- 7"; volume: "49"; issue: "8";
            pages: "4793-4802"
COMMENT     Sequence Label: pJEC625
FEATURES             Location/Qualifiers
     source          1..2585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(250..1041,1042..1110))
                     /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     873..892
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1111..1215)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1283..1301
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1305..1348)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        1366..1400
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1422..1497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1518..1547
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1563..1580
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1636..1653)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1806..1852
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1905..1922)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2076..2585,1..79))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2156..2175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
       61 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      121 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      181 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      241 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      301 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      361 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      421 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
      481 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
      541 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
      601 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
      661 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
      721 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
      781 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
      841 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
      901 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
      961 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1021 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1081 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1141 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1201 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     1261 tatcatgaca ttaacctata aaaataggcg tatcacgagg cagaatttca gataaaaaaa
     1321 atccttagct ttcgctaagg atgatttctg gaattctaaa gatctttgac agctagctca
     1381 gtcctaggta taatactagt tagtcttcga taagaagact agttttagag ctagaaatag
     1441 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     1501 ttttgaagct tgggcccgaa caaaaactca tctcagaaga ggatctgaat agcgccgtcg
     1561 accatcatca tcatcatcat tgagtttaaa cggtctccag cttggctgtt ttggcggatg
     1621 agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca
     1681 gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     1741 gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
     1801 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     1861 tgtcggtgaa ctggatcctt actcgagtct agactgcagg cttcctcgct cactgactcg
     1921 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1981 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2041 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc acaggctccg cccccctgac
     2101 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2161 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2221 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2281 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2341 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2401 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2461 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2521 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2581 tgatc
//
