LOCUS       sequence-211104-        7112 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 7112)
  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 7112)
  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..7112
                     /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      1291..1879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_RNA        2093..2111
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2163..2181
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     enhancer        2309..2594
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2596..2873
                     /label=chicken beta-actin promoter
     intron          2874..3102
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3122..3508
                     /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             3563..3583
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4153..4170)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4159..4366
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4375..4515
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4375..4504
                     /label=AAV2 ITR
     rep_origin      4590..5045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4677..4696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4887..4908
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5062..5081)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5181..5203
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5327..5431
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5432..5500,5501..6292)
                     /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      6463..7051
                     /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 ttttgcccaa
      901 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagtaatttg
      961 gagccggtga gcgtggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1021 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1081 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1141 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1201 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1261 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1321 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1381 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1441 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1501 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1561 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1621 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1681 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1741 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1801 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1861 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1921 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     1981 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2041 agtgagcgag gaagcggaag agcgcgagaa gacctgtttt agagctaggc caacatgagg
     2101 atcacccatg tctgcagggc ctagcaagtt aaaataaggc tagtccgtta tcaacttggc
     2161 caacatgagg atcacccatg tctgcagggc caagtggcac cgagtcggtg ctttttttgt
     2221 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
     2281 tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg
     2341 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
     2401 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     2461 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     2521 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     2581 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     2641 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     2701 cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
     2761 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     2821 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     2881 ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     2941 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     3001 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
     3061 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtccaccat
     3121 ggcttcaaac tttactcagt tcgtgctcgt ggacaatggt gggacagggg atgtgacagt
     3181 ggctccttct aatttcgcta atggggtggc agagtggatc agctccaact cacggagcca
     3241 ggcctacaag gtgacatgca gcgtcaggca gtctagtgcc cagaagagaa agtataccat
     3301 caaggtggag gtccccaaag tggctaccca gacagtgggc ggagtcgaac tgcctgtcgc
     3361 cgcttggagg tcctacctga acatggagct cactatccca attttcgcta ccaattctga
     3421 ctgtgaactc atcgtgaagg caatgcaggg gctcctcaaa gacggtaatc ctatcccttc
     3481 cgccatcgcc gctaactcag gtatctacag cgctggagga ggtggaagcg gaggaggagg
     3541 aagcggagga ggaggtagcg gacctaagaa aaagaggaag gtggcggccg ctggaatgga
     3601 ccccgctacc ttcacatacc aattcaaaaa tgtccgctgg gctaagggtc ggcgtgagac
     3661 ctacctgtgc tacgtagtga agaggcgtga cagtgctaca tccttttcac tggactttgg
     3721 ttatcttcgc aataagaacg gctgccacgt ggaattgctc ttcctccgct acatctcgga
     3781 ctgggaccta gaccctggcc gctgctaccg cgtcacctgg ttcacctcct ggagcccctg
     3841 ctacgactgt gcccgacatg tggccgactt tctgcgaggg aaccccaacc tcagtctgag
     3901 gatcttcacc gcgcgcctct actacttcca atacccctgc taccaagagg ggctgcggcg
     3961 gctgcaccgc gccggggtgc aaatagccat catgaccttc aaagattatt tttactgctg
     4021 gaatactttt gtagaaaacc atgaaagaac tttcaaagcc tgggaagggc tgcatgaaaa
     4081 ttcagttcgt ctctccagac agcttcggcg catccttcag taagatccta attcctagag
     4141 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4201 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4261 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4321 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     4381 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     4441 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     4501 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4561 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     4621 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4681 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4741 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4801 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4861 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4921 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4981 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     5041 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     5101 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5161 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5221 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5281 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5341 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5401 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5461 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5521 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5581 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5641 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5701 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5761 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5821 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5881 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5941 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6001 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6061 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6121 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     6181 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6241 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6301 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6361 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6421 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6481 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6541 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6601 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6661 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6721 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6781 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6841 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6901 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6961 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7021 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7081 cctggccttt tgctggcctt ttgctcacat gt
//
