LOCUS       sequence-170754-        2709 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template 2 (of 2) for U6>sgRNA cassette OSO-PCR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2709)
  AUTHORS   Gandhi S, Haeussler M, Razy-Krajka F, Christiaen L, Stolfi A
  TITLE     Evaluation and rational design of guide RNAs for efficient 
            CRISPR/Cas9-mediated mutagenesis in Ciona.
  JOURNAL   Dev Biol. 2017 May 1;425(1):8-20. doi: 10.1016/j.ydbio.2017.03.003. 
            Epub 2017 Mar 22.
  PUBMED    28341547
REFERENCE   2  (bases 1 to 2709)
  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: 
            "10.1016/j.ydbio.2017.03.003"; journalName: "Dev Biol"; date: 
            "2017-05-1- 1"; volume: "425"; issue: "1"; pages: "8-20"
COMMENT     Sequence Label: XX>sgRNA F+E
FEATURES             Location/Qualifiers
     source          1..2709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    456..590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(493..512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     547..566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(626..645)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(627..645)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(732..749)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(903..1491)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(983..1002)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1662..2453,2454..2522))
                     /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     2285..2304
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2523..2627)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(2695..2709,1..4)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aggcccttac gtattaatta aggcgcgccc gactcgtgcg aagtttaaga gctatgctgg
       61 aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      121 gtcggtgctt tttttgaatt ccagctgagc gccggtcgct accattacca gttggtctgg
      181 tgtcaaaaat aataataacc gggcaggcca tgtctgcccg tatttcgcgt aaggaaatcc
      241 attatgtact atttaaaaaa cacaaacttt tggatgttcg gtttattctt tttcttttac
      301 ttttttatca tgggagccta cttcccgttt ttcccgattt ggctacatga catcaaccat
      361 atcagcaaaa gtgatacggg tattattttt gccgctattt ctctgttctc gctattattc
      421 caaccgctgt ttggtctgct ttctgacaaa ctcggaactt gtttattgca gcttataatg
      481 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
      541 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gacaaagtca
      601 aagcggccat cagatctgcc ggactcccta tagtgagtcg tattaatttc gataagccag
      661 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
      721 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
      781 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      841 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      901 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      961 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1021 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1081 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1141 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1201 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1261 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1321 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     1381 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1441 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1501 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1561 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1621 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1681 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1741 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1801 cgagatccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1861 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1921 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1981 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2041 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2101 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2161 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2221 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2281 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2341 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2401 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2461 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2521 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2581 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     2641 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     2701 cgtatcacg
//
