LOCUS       sequence-211106-        7184 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  empty sgRNA cloning vector with MS2-humanA3a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7184)
  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 7184)
  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: pLL4 sgRNA(MS2)-MS2-hA3a
FEATURES             Location/Qualifiers
     source          1..7184
                     /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"
     CDS             3596..4195
                     /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
                     GCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWD
                     TFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN"
     primer_bind     complement(4225..4242)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4231..4438
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4447..4587
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4447..4576
                     /label=AAV2 ITR
     rep_origin      4662..5117
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4749..4768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4959..4980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5134..5153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5253..5275
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5399..5503
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5504..5572,5573..6364)
                     /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      6535..7123
                     /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 agccagccca gcatccgggc ccagacactt gatggatcca cacatattca cttccaactt
     3661 taacaatggc attggaaggc ataagaccta cctgtgctac gaagtggagc gcctggacaa
     3721 tggcacctcg gtcaagatgg accagcacag gggctttcta cacaaccagg ctaagaatct
     3781 tctctgtggc ttttacggcc gccatgcgga gctgcgcttc ttggacctgg ttccttcttt
     3841 gcagttggac ccggcccaga tctacagggt cacttggttc atctcctgga gcccctgctt
     3901 ctcctggggc tgtgccgggg aagtgcgtgc gttccttcag gagaacacac acgtgagact
     3961 gcgtatcttc gctgcccgca tctatgatta cgacccccta tataaggagg cactgcaaat
     4021 gctgcgggat gctggggccc aagtctccat catgacctac gatgaattta agcactgctg
     4081 ggacaccttt gtggaccacc agggatgtcc cttccagccc tgggatggac tagatgagca
     4141 cagccaagcc ctgagtggga ggctgcgggc cattctccag aatcagggaa actgagatcc
     4201 taattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     4261 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     4321 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     4381 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc
     4441 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     4501 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     4561 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     4621 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     4681 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4741 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4801 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4861 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4921 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4981 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5041 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5101 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5161 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5221 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5281 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5341 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5401 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5461 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5521 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5581 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5641 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5701 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5761 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5821 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5881 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5941 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6001 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6061 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6121 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6181 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     6241 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6301 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6361 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6421 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6481 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6541 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6601 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6661 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     6721 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6781 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6841 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6901 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6961 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7021 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7081 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7141 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
