LOCUS       sequence-222674-        7182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty sgRNA cloning vector with MS2-humanA3a D131E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7182)
  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 7182)
  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: pLL6 sgRNA(MS2)-MS2-hA3a D131E
FEATURES             Location/Qualifiers
     source          1..7182
                     /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"
     CDS             3594..4193
                     /codon_start=1
                     /gene="human APOBEC3A"
                     /gene_synonym="A3A"
                     /product="human cytidine deaminase (C to U editing enzyme) 
                     that efficiently edits methylated cytosines (Wang et al., 
                     2018)"
                     /label=APOBEC3A
                     /note="also known as hA3A"
                     /translation="MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTS
                     VKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW
                     GCAGEVRAFLQENTHVRLRIFAARIYEYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWD
                     TFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN"
     primer_bind     complement(4223..4240)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4229..4436
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4445..4585
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4445..4574
                     /label=AAV2 ITR
     rep_origin      4660..5115
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4747..4766)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4957..4978
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5132..5151)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5251..5273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5397..5501
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5502..5570,5571..6362)
                     /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      6533..7121
                     /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 ggaatggaag
     3601 ccagcccagc atccgggccc agacacttga tggatccaca catattcact tccaacttta
     3661 acaatggcat tggaaggcat aagacctacc tgtgctacga agtggagcgc ctggacaatg
     3721 gcacctcggt caagatggac cagcacaggg gctttctaca caaccaggct aagaatcttc
     3781 tctgtggctt ttacggccgc catgcggagc tgcgcttctt ggacctggtt ccttctttgc
     3841 agttggaccc ggcccagatc tacagggtca cttggttcat ctcctggagc ccctgcttct
     3901 cctggggctg tgccggggaa gtgcgtgcgt tccttcagga gaacacacac gtgagactgc
     3961 gtatcttcgc tgcccgcatc tatgagtacg accccctata taaggaggca ctgcaaatgc
     4021 tgcgggatgc tggggcccaa gtctccatca tgacctacga tgaatttaag cactgctggg
     4081 acacctttgt ggaccaccag ggatgtccct tccagccctg ggatggacta gatgagcaca
     4141 gccaagccct gagtgggagg ctgcgggcca ttctccagaa tcagggaaac tgagatccta
     4201 attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     4261 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     4321 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     4381 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg
     4441 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4501 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4561 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4621 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4681 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     4741 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4801 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4861 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4921 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4981 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     5041 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5101 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5161 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5221 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5281 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5341 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5401 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5461 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5521 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5581 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5641 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5701 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5761 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5821 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5881 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5941 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6001 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6061 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6121 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6181 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6241 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6301 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6361 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7141 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
