LOCUS       sequence-169993-        5474 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  minicircle parental backbone plasmid including GFPNLS-pA knock-in 
            donor and one cutting site for HITI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5474)
  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 5474)
  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..5474
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(702..719)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    740..773
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     regulatory      803..812
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(809..811,812..814,815..1525)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(809..811,812..814,815..1525)
                     /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(854..875)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1115..1134)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1462..1483
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1544..1564
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1568..1588
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1592..1612
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1686..1705)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1751..1806
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1805..1824)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2449..2468)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3061..3080
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(3497..3514)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3667..3753
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3845..3872
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4006..4594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4495..4514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4748..4765
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4780..4920
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4846..4865)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5006..5028
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acattaccct gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt
       61 atccctagat gacattaccc tgttatccct agatgacatt taccctgtta tccctagatg
      121 acattaccct gttatcccag atgacattac cctgttatcc ctagatacat taccctgtta
      181 tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt
      241 accctgttat ccctagatac attaccctgt tatcccagat gacataccct gttatcccta
      301 gatgacatta ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct
      361 gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta tcccagatga
      421 cattaccctg ttatccctag atacattacc ctgttatccc agatgacata ccctgttatc
      481 cctagatgac attaccctgt tatcccagat gacattaccc tgttatccct agatacatta
      541 ccctgttatc ccagatgaca taccctgtta tccctagatg acattaccct gttatcccag
      601 atgacattac cctgttatcc ctagatacat taccctgtta tcccagatga cataccctgt
      661 tatccctaga tgacattacc ctgttatccc agataaactc aatgatgatg atgatgatgg
      721 tcgagactca gcggccgcgg tgccagggcg tgcccttggg ctccccgggc gcgactatag
      781 aggaacttct tagggcccgc gggccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      841 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      901 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      961 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     1021 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     1081 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     1141 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     1201 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1261 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1321 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1381 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     1441 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1501 tctcggcatg gacgagctgt acaagtccgg actcagatct gatccaaaaa agaagagaaa
     1561 ggtagatcca aaaaagaaga gaaaggtaga tccaaaaaag aagagaaagg tataaaagct
     1621 tcgaggaatt cgaagcttct aggtaccaga tatcaggatc ctggccagct agctaggtag
     1681 ctagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     1741 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     1801 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     1861 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     1921 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     1981 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     2041 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     2101 gtcatagctg tccctcttct cttatggaga tccctcgacc gatccgctgc attaatgaat
     2161 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttcgggcc cgccccaact
     2221 ggggtaacct ttgagttctc tcagttgggg gtaatcagca tcatgatgtg gtaccacatc
     2281 atgatgctga ttataagaat gcggccgcca cactctagtg gatctcgagt taataattca
     2341 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     2401 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     2461 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     2521 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     2581 gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg ctggcgcgag
     2641 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     2701 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     2761 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgtag
     2821 atgacatgga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     2881 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     2941 gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     3001 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     3061 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     3121 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagagct tgatcccctg
     3181 cgccatcaga tccttggcgg cgagaaagcc atccagttta ctttgcaggg cttcccaacc
     3241 ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag
     3301 tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt
     3361 tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga
     3421 ctggctttct acgtgctcga ggggggccaa acggtctcca gcttggctgt tttggcggat
     3481 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     3541 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     3601 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     3661 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     3721 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     3781 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     3841 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     3901 tttctaaata cattcaaata tgtatccgct catgaccaaa atcccttaac gtgagttttc
     3961 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4021 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4081 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4141 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4201 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4261 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4321 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4381 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4441 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4501 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4561 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4621 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4681 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4741 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     4801 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     4861 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4921 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4981 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5041 tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa
     5101 tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg
     5161 tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct
     5221 tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag
     5281 aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg
     5341 gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta
     5401 atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt
     5461 gcgacgctgg cgat
//
