LOCUS       sequence-174925-        6092 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  minicircle parental backbone plasmid including luc-pA knock-in donor
            and one cutting site for HITI .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 6092)
  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 6092)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-luc-MC
FEATURES             Location/Qualifiers
     source          1..6092
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(740..757)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    778..811
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     regulatory      841..850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             847..2496
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    2551..2672
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2562..2581)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2616..2635
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2957..3751)
                     /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(2957..3751)
                     /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(3068..3087)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3678..3697
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4115..4132)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4285..4371
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4463..4490
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4624..5212
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5113..5132
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5366..5383
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5398..5538
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5464..5483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5624..5646
                     /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 acatnaccct gttatcccta gatnacatta ccctgttatc ccagatgaca taccctgtta
      241 tcccyagatg acattaccct gttatcccag atgacattac cctgttatcc ctagatacat
      301 taccctgtta tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc
      361 agatgacatt accctgttat ccctagatac attaccctgt tatcccagat gacataccct
      421 gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt atccctagat
      481 acattaccct gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta
      541 tcccagatga cattaccctg ttatccctag atacattacc ctgttatccc agatgacata
      601 ccctgttatc cctagatgac attaccctgt tatcccagat gacattaccc tgttatccct
      661 agatacatta ccctgttatc ccagatgaca taccctgtta tccctagatg acattaccct
      721 gttatcccag ataaactcaa tgatgatgat gatgatggtc gagactcagc ggccgcggtg
      781 ccagggcgtg cccttgggct ccccgggcgc gactatagag gaacttctta gggcccgcgg
      841 gccaccatgg aagatgccaa aaacattaag aagggcccag cgccattcta cccactcgaa
      901 gacgggaccg ccggcgagca gctgcacaaa gccatgaagc gctacgccct ggtgcccggc
      961 accatcgcct ttaccgacgc acatatcgag gtggacatta cctacgccga gtacttcgag
     1021 atgagcgttc ggctggcaga agctatgaag cgctatgggc tgaatacaaa ccatcggatc
     1081 gtggtgtgca gcgagaatag cttgcagttc ttcatgcccg tgttgggtgc cctgttcatc
     1141 ggtgtggctg tggccccagc taacgacatc tacaacgagc gcgagctgct gaacagcatg
     1201 ggcatcagcc agcccaccgt cgtattcgtg agcaagaaag ggctgcaaaa gatcctcaac
     1261 gtgcaaaaga agctaccgat catacaaaag atcatcatca tggatagcaa gaccgactac
     1321 cagggcttcc aaagcatgta caccttcgtg acttcccatt tgccacccgg cttcaacgag
     1381 tacgacttcg tgcccgagag cttcgaccgg gacaaaacca tcgccctgat catgaacagt
     1441 agtggcagta ccggattgcc caagggcgta gccctaccgc accgcaccgc ttgtgtccga
     1501 ttcagtcatg cccgcgaccc catcttcggc aaccagatca tccccgacac cgctatcctc
     1561 agcgtggtgc catttcacca cggcttcggc atgttcacca cgctgggcta cttgatctgc
     1621 ggctttcggg tcgtgctcat gtaccgcttc gaggaggagc tattcttgcg cagcttgcaa
     1681 gactataaga ttcaatctgc cctgctggtg cccacactat ttagcttctt cgctaagagc
     1741 actctcatcg acaagtacga cctaagcaac ttgcacgaga tcgccagcgg cggggcgccg
     1801 ctcagcaagg aggtaggtga ggccgtggcc aaacgcttcc acctaccagg catccgccag
     1861 ggctacggcc tgacagaaac aaccagcgcc attctgatca cccccgaagg ggacgacaag
     1921 cctggcgcag taggcaaggt ggtgcccttc ttcgaggcta aggtggtgga cttggacacc
     1981 ggtaagacac tgggtgtgaa ccagcgcggc gagctgtgcg tccgtggccc catgatcatg
     2041 agcggctacg ttaacaaccc cgaggctaca aacgctctca tcgacaagga cggctggctg
     2101 cacagcggcg acatcgccta ctgggacgag gacgagcact tcttcatcgt ggaccggctg
     2161 aagagcctga tcaaatacaa gggctaccag gtagccccag ccgaactgga gagcatcctg
     2221 ctgcaacacc ccaacatctt cgacgccggg gtcgccggcc tgcccgacga cgatgccggc
     2281 gagctgcccg ccgcagtcgt cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc
     2341 gtggactatg tggccagcca ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc
     2401 gtggacgagg tgcctaaagg actgaccggc aagttggacg cccgcaagat ccgcgagatt
     2461 ctcattaagg ccaagaaggg cggcaagatc gccgtgaatt cttaactgca gtctagagtc
     2521 ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     2581 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2641 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2701 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     2761 gtaaaatcga taaggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2821 ttccgggccc gccccaactg gggtaacctt tgagttctct cagttggggg taatcagcat
     2881 catgatgtgg taccacatca tgatgctgat tataagaatg cggccgccac actctagtgg
     2941 atctcgagtt aataattcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
     3001 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
     3061 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
     3121 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
     3181 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga
     3241 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
     3301 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
     3361 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
     3421 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
     3481 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
     3541 ccagccacga tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg
     3601 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
     3661 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     3721 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     3781 cagagcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     3841 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg
     3901 tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc
     3961 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     4021 agcaccgttt ctgcggactg gctttctacg cgctcgaggg ggggccaaac ggtctccagc
     4081 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     4141 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     4201 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     4261 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4321 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     4381 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     4441 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     4501 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaccaaaat
     4561 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4621 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4681 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4741 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     4801 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4861 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4921 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4981 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5041 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5101 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5161 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5221 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5281 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     5341 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     5401 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     5461 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     5521 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5581 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5641 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag
     5701 gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta
     5761 tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct
     5821 acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat
     5881 tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg
     5941 gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg
     6001 cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc
     6061 gacaagcaaa catgctgtgc gacgctggcg at
//
