LOCUS       sequence-161640-        2789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-based cloning of SpCas9 gRNA guide sequence, position 7/17/27 
            in the array.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2789)
  AUTHORS   Vad-Nielsen J, Lin L, Bolund L, Nielsen AL, Luo Y
  TITLE     Golden Gate Assembly of CRISPR gRNA expression array for 
            simultaneously targeting multiple genes.
  JOURNAL   Cell Mol Life Sci. 2016 May 13.
  PUBMED    27178736
REFERENCE   2  (bases 1 to 2789)
  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: "Cell Mol 
            Life Sci. 2016 May 13."
COMMENT     Sequence Label: pMA-SpCas9-g7
FEATURES             Location/Qualifiers
     source          1..2789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     438..457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     630..647
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_RNA        complement(834..909)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(928..1176)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(986..1005)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1156..1176)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1240..1259)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1241..1259)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1270..1287)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1270..1286)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1279..1301)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1602..1706
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1707..1775,1776..2567)
                     /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(1925..1944)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2738..2789,1..537)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
       61 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      121 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      181 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      241 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      301 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      361 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      421 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      481 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      541 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcattaggca
      601 ccccaggctt tacccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggagagcg
      661 cccaatacgc aaggaaacag ctatgaccat gttaatgcag ctggcacgac aggtttcccg
      721 actggaaagc gggcagtgag cggaaggccc gtgaggccag tcttgtgctc caggtaccac
      781 gcgttgtaca ggtctcgttt ctaacttgct atttctagct ctaaaacaaa aaagcaccga
      841 ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatttctag
      901 ctctaaaaca ggtcttctcg aagacccggt gtttcgtcct ttccacaaga tatataaagc
      961 caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa
     1021 ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa
     1081 tatctttgtg tttacagtca aattaattcc aattatctct ctaacagcct tgtatcgtat
     1141 atgcaaatat gaaggaatca tgggaaatag gccctcaccg tgagaccgga tccgagctct
     1201 ggacaagaca cgtggccttg acggccttcc gccaattcgc cctatagtga gtcgtattac
     1261 gtcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1321 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1381 caccgaaacg cccttcccaa cagttgcgca gcctgaatgg cgaatgggag cgccctgtag
     1441 cggccactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     1501 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     1561 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1621 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     1681 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     1741 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     1801 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     1861 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     1921 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     1981 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2041 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     2101 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     2161 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     2221 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     2281 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     2341 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     2401 aatctggagc cggtgagcgt ggttctcgcg gtatcattgc agcactgggg ccagatggta
     2461 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     2521 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     2581 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     2641 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2701 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2761 taatctgctg cttgcaaaca aaaaaacca
//
