LOCUS       sequence-189860-        3018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty gRNA scaffold with ""Flip"" and ""Extension"" modifications 
            with optimized chicken specific U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3018)
  AUTHORS   Gandhi S, Piacentino ML, Vieceli FM, Bronner ME
  TITLE     Optimization of CRISPR/Cas9 genome editing for loss-of-function in 
            the early chick embryo.
  JOURNAL   Dev Biol. 2017 Dec 1;432(1):86-97. doi: 10.1016/j.ydbio.2017.08.036.
  PUBMED    29150011
REFERENCE   2  (bases 1 to 3018)
  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.2017.08"; journalName: "Dev Biol"; date: 
            "2017-12-1- 1"; volume: "432"; issue: "1"; pages: "86-97"
COMMENT     Sequence Label: U6.3>gRNA.f+e
FEATURES             Location/Qualifiers
     source          1..3018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    736..870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(773..792)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     827..846
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(906..925)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(907..925)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1012..1029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1183..1771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1263..1282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1942..2733,2734..2802))
                     /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     2565..2584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2803..2907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2975..2993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gcgccgccgc tccttcaggc actgccgacg acagcccagg cggaggtcct gagcgccggc
       61 gctaaatttg cataaagaac tacccaggag ccctcgcgcg cggaaacggg caaaaagggg
      121 cttctaatat ggaaatatta cgccgaatcg cgttacaaat cggctaagcg ggcctaagag
      181 ttaacaagat gtgctattaa gcggagcctt ttggtgggaa gaaatggagt agtcactgtg
      241 ttctaaaaga acttgcagaa tgagccttta aataccgcag tctcgatgct cttagtcgga
      301 tagagaccga gagagggtct cagtttaaga gctatgctgg aaacagcata gcaagtttaa
      361 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaatt
      421 ccagctgagc gccggtcgct accattacca gttggtctgg tgtcaaaaat aataataacc
      481 gggcaggcca tgtctgcccg tatttcgcgt aaggaaatcc attatgtact atttaaaaaa
      541 cacaaacttt tggatgttcg gtttattctt tttcttttac ttttttatca tgggagccta
      601 cttcccgttt ttcccgattt ggctacatga catcaaccat atcagcaaaa gtgatacggg
      661 tattattttt gccgctattt ctctgttctc gctattattc caaccgctgt ttggtctgct
      721 ttctgacaaa ctcggaactt gtttattgca gcttataatg gttacaaata aagcaatagc
      781 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
      841 ctcatcaatg tatcttatca tgtctggatc gacaaagtca aagcggccat cagatctgcc
      901 ggactcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
      961 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     1021 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1081 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1141 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1201 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1261 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1321 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1381 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1441 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1501 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1561 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1621 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1681 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1741 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     1801 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     1861 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     1921 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     1981 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2041 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagatccac gctcaccggc
     2101 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2161 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2221 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2281 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2341 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2401 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2461 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2521 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2581 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2641 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2701 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     2761 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     2821 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2881 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     2941 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggcccttacg
     3001 tattaattaa ggcgcgcc
//
