LOCUS       sequence-222656-        7110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty sgRNA cloning vector with MS2-AID3c.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7110)
  AUTHORS   Liu LD, Huang M, Dai P, Liu T, Fan S, Cheng X, Zhao Y, Yeap LS, Meng
            FL
  TITLE     Intrinsic Nucleotide Preference of Diversifying Base Editors Guides 
            Antibody Ex Vivo Affinity Maturation.
  JOURNAL   Cell Rep. 2018 Oct 23;25(4):884-892.e3. doi: 
            10.1016/j.celrep.2018.09.090.
  PUBMED    30355495
REFERENCE   2  (bases 1 to 7110)
  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.celrep.2018.09"; journalName: "Cell Rep"; date: 
            "2018-10-23- 23"; volume: "25"; issue: "4"; pages: "884-892.e3"
COMMENT     Sequence Label: pLL2 sgRNA(MS2)-MS2-AID3c
FEATURES             Location/Qualifiers
     source          1..7110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        341..445
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1292..1880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_RNA        2092..2110
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2162..2180
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     enhancer        2308..2593
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2595..2872
                     /label=chicken beta-actin promoter
     intron          2873..3100
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3120..3506
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             3561..3581
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4151..4168)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4157..4364
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4373..4513
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4373..4502
                     /label=AAV2 ITR
     rep_origin      4588..5043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4675..4694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4885..4906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5060..5079)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5179..5201
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5325..5429
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5430..5498,5499..6290)
                     /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"
     rep_origin      6461..7049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttctcg tgatacgcct atttttatag gttaatgtca tgataataat
      301 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
      361 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
      421 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
      481 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      541 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
      601 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
      661 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
      721 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
      781 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
      841 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
      901 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagcaatctg
      961 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     1021 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1081 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1141 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1201 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1261 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1321 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1381 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1441 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1501 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1561 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1621 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1681 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1741 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1801 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1861 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1921 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1981 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     2041 cagtgagcga ggaagcggaa gagcgcgaag acctgtttta gagctaggcc aacatgagga
     2101 tcacccatgt ctgcagggcc tagcaagtta aaataaggct agtccgttat caacttggcc
     2161 aacatgagga tcacccatgt ctgcagggcc aagtggcacc gagtcggtgc tttttttgtt
     2221 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt
     2281 ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg
     2341 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca
     2401 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     2461 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg
     2521 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     2581 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
     2641 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
     2701 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
     2761 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
     2821 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     2881 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     2941 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     3001 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     3061 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tccaccatgg
     3121 cttcaaactt tactcagttc gtgctcgtgg acaatggtgg gacaggggat gtgacagtgg
     3181 ctccttctaa tttcgctaat ggggtggcag agtggatcag ctccaactca cggagccagg
     3241 cctacaaggt gacatgcagc gtcaggcagt ctagtgccca gaagagaaag tataccatca
     3301 aggtggaggt ccccaaagtg gctacccaga cagtgggcgg agtcgaactg cctgtcgccg
     3361 cttggaggtc ctacctgaac atggagctca ctatcccaat tttcgctacc aattctgact
     3421 gtgaactcat cgtgaaggca atgcaggggc tcctcaaaga cggtaatcct atcccttccg
     3481 ccatcgccgc taactcaggt atctacagcg ctggaggagg tggaagcgga ggaggaggaa
     3541 gcggaggagg aggtagcgga cctaagaaaa agaggaaggt ggcggccgct ggaatggacc
     3601 ccgctacctt cacataccaa ttcaaaaatg tccgctgggc taagggtcgg cgtgagacct
     3661 acctgtgcta cgtagtgaag aggcgtgaca gtgctacatc cttttcactg gactttggtt
     3721 atcttcgcaa taagaacggc tgccacgtgg aattgctctt cctccgctac atctcggact
     3781 gggacctaga ccctggccgc tgctaccgcg tcacctggtt cacctcctgg agcccctgct
     3841 acgactgtgc ccgacatgtg gccgactttc tgcgagggaa ccccaacctc agtctgagga
     3901 tcttcaccgc gcgcctctac tacttccaat acccctgcta ccaagagggg ctgcggcggc
     3961 tgcaccgcgc cggggtgcaa atagccatca tgaccttcaa agattatttt tactgctgga
     4021 atacttttgt agaaaaccat gaaagaactt tcaaagcctg ggaagggctg catgaaaatt
     4081 cagttcgtct ctccagacag cttcggcgca tccttcagta agatcctaat tcctagagct
     4141 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4201 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4261 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4321 agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc
     4381 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4441 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4501 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4561 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4621 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4681 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4741 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4801 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4861 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     4921 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     4981 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5041 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5101 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5161 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5221 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5281 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5341 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5401 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5461 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5521 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5581 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5641 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5701 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5761 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5821 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     5881 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     5941 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6001 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6061 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6121 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6181 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6241 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6301 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6361 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6421 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6481 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6541 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6601 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6661 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6721 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6781 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6841 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6901 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6961 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7021 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7081 tggccttttg ctggcctttt gctcacatgt
//
