LOCUS       sequence-222647-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty sgRNA cloning vector with MS2-AIDmono.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  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 7113)
  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: pLL1 sgRNA(MS2)-MS2-AIDmono
FEATURES             Location/Qualifiers
     source          1..7113
                     /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(4154..4171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4160..4367
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4376..4516
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4376..4505
                     /label=AAV2 ITR
     rep_origin      4591..5046
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4678..4697)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4888..4909
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5063..5082)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5182..5204
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5328..5432
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5433..5501,5502..6293)
                     /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      6464..7052
                     /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 aakaaatctg
      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 ttctgtgagg accgcaaggc tgagcccgag gggctgcggc
     3961 ggctggcaga agccggggtg caaatagcca tcatgacctt caaagattat ttttactgct
     4021 ggaatacttt tgtagaaaac catgaaagaa ctttcaaagc ctgggaaggg ctgcatgaaa
     4081 attcagttcg tctctccaga cagcttcggc gcatccttca gtaagatcct aattcctaga
     4141 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4201 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4261 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4321 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa
     4381 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     4441 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     4501 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4561 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4621 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     4681 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4741 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4801 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     4861 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     4921 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     4981 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     5041 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5101 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5161 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5221 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5281 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5341 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5401 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5461 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5521 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5581 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5641 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5701 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5761 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5821 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5881 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5941 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6001 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6061 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6121 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6181 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6241 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6301 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6361 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6421 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6481 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6541 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6601 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6661 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6721 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6781 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6841 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6901 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6961 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7021 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7081 tcctggcctt ttgctggcct tttgctcaca tgt
//
