LOCUS       sequence-174927-        5510 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  minicircle parental backbone plasmid including GFPNLS-pA knock-in 
            donor and one cutting site for HITI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5510)
  AUTHORS   Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, 
            Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, 
            Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
            Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, 
            Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang 
            K, Callaway EM, Zhang K, Belmonte JC
  TITLE     In vivo genome editing via CRISPR/Cas9 mediated homology-independent
            targeted integration.
  JOURNAL   Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
            2016 Nov 16.
  PUBMED    27851729
REFERENCE   2  (bases 1 to 5510)
  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: 
            "10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1"; 
            volume: "540"; issue: "7631"; pages: "144-149"
COMMENT     Sequence Label: pAi14-GFPNLS-MC
FEATURES             Location/Qualifiers
     source          1..5510
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(739..756)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    777..810
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     regulatory      840..849
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(846..848,849..851,852..1562)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(846..848,849..851,852..1562)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(891..912)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1152..1171)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1499..1520
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1581..1601
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1605..1625
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1629..1649
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1723..1742)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1788..1843
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1842..1861)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     CDS             complement(2375..3169)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2375..3169)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2486..2505)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3096..3115
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(3533..3550)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3703..3789
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3881..3908
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4042..4630
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4531..4550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4784..4801
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4816..4956
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4882..4901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5042..5064
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcttctctca tccgccaaaa cagccaagct ggagaccgtt tgacattacc ctgttatccc
       61 tagatacatt accctgttat cccagatgac ataccctgtt atccctagat gacattaccc
      121 tgttatccca gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg
      181 acataccctg ttatccctag atgacattac cctgttatcc cagatgacat acccygttat
      241 cccyagatga cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt
      301 accctgttat cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca
      361 gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg
      421 ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta tccctagata
      481 cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt accctgttat
      541 cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca gatgacatac
      601 cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct gttatcccta
      661 gatacattac cctgttatcc cagatgacat accctgttat ccctagatga cattaccctg
      721 ttatcccaga taaactcaat gatgatgatg atgatggtcg agactcagcg gccgcggtgc
      781 cagggcgtgc ccttgggctc cccgggcgcg actatagagg aacttcttag ggcccgcggg
      841 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      901 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      961 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1021 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1081 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1141 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1201 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1261 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1321 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1381 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1441 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1501 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1561 agtccggact cagatctgat ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa
     1621 aggtagatcc aaaaaagaag agaaaggtat aaaagcttcg aggaattcga agcttctagg
     1681 taccagatat caggatcctg gccagctagc taggtagcta gatctttttc cctctgccaa
     1741 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     1801 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     1861 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     1921 ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca
     1981 gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt
     2041 tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt
     2101 actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt
     2161 atggagatcc ctcgaccgat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2221 gtttgcgtat tgggcgctct tcgggcccgc cccaactggg gtaacctttg agttctctca
     2281 gttgggggta atcagcatca tgatgtggta ccacatcatg atgctgatta taagaatgcg
     2341 gccgccacac tctagtggat ctcgagttaa taattcagaa gaactcgtca agaaggcgat
     2401 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag
     2461 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc
     2521 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca
     2581 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc
     2641 tcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat
     2701 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg
     2761 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag
     2821 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca
     2881 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc
     2941 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc agttcattca
     3001 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga
     3061 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct
     3121 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc
     3181 ctcatcctgt ctcttgatca gagcttgatc ccctgcgcca tcagatcctt ggcggcaaga
     3241 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca
     3301 attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac
     3361 tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc
     3421 tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgcg ctcgaggggg
     3481 ggccaaacgg tctccagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
     3541 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     3601 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     3661 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     3721 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     3781 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     3841 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     3901 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     3961 tccgctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4021 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4081 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4141 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4201 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4261 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4321 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4381 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4441 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4501 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4561 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4621 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4681 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4741 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4801 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4861 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4921 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4981 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5041 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag
     5101 atcaattcgc gcgcgaaggc gaagcggcat gcataatgtg cctgtcaaat ggacgaagca
     5161 gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat
     5221 tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc
     5281 gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca
     5341 acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg
     5401 ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga
     5461 tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat
//
