LOCUS       sequence-240498-        5768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology repair template for in frame (first exon) clover knock-in 
            of human LMNA gene .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5768)
  AUTHORS   Pinder J, Salsman J, Dellaire G
  TITLE     Nuclear domain 'knock-in' screen for the evaluation and 
            identification of small molecule enhancers of CRISPR-based genome 
            editing.
  JOURNAL   Nucleic Acids Res. 2015 Oct 30;43(19):9379-92. doi: 
            10.1093/nar/gkv993. Epub 2015 Oct 1.
  PUBMED    26429972
REFERENCE   2  (bases 1 to 5768)
  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: 
            "10.1093/nar/gkv993"; journalName: "Nucleic Acids Res"; date: 
            "2015-10-30- 30"; volume: "43"; issue: "19"; pages: "9379-92"
COMMENT     Sequence Label: pCR2.1 Clover-LMNA Donor
FEATURES             Location/Qualifiers
     source          1..5768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(56..58,59..61,62..64,65..775)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MEVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLT
                     LKFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKD
                     DGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGI
                     KANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVL
                     LEFVTAAGITHGMDELYK"
     primer_bind     complement(104..125)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     712..733
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1509..1528)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1510..1528)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1535..1552)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1535..1551)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1544..1566)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1692..2120
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1779..1798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1989..2010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2464..3258
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2518..2537)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3128..3147
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(3276..3344,3345..4136)
                     /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"
     primer_bind     complement(3494..3513)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4307..4895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4796..4815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5049..5066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5183..5204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5219..5224,5225..5242,5243..5249)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5257..5273
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5262..5284
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5281..5297
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5281..5297
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cccgcgccct ttccgggacc cctgccccgc gggcagcgct gccaacctgc cggccatgga
       61 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      121 cgacgtaaac ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg
      181 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      241 cgtgaccacc ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca
      301 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt
      361 caaggacgac ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      421 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      481 gctggagtac aacttcaaca gccacaacgt ctatatcacg gccgacaagc agaagaacgg
      541 catcaaggct aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga
      601 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
      661 cctgagccat cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
      721 gctggagttc gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagacccc
      781 gtcccagcgg cgcgccaccc gcagcggggc gcaggccagc tccactccgc tgtcgcccac
      841 ccgcatcacc cggctgcagg agaaggagga cctgcaggag ctcaatgatc gcttggcggt
      901 ctacatcgac cgtgtgcgct cgctggaaac ggagaacgca gggctgcgcc ttcgcatcac
      961 cgagtctgaa gaggtggtca gccgcgaggt gtccggcatc aaggccgcct acgaggccga
     1021 gctcggggat gcccgcaaga cccttgactc agtagccaag gagcgcgccc gcctgcagct
     1081 ggagctgagc aaagtgcgtg aggagtttaa ggagctgaaa gcgcggtgag ttcgcccagg
     1141 tggctgcgtg cctggcgggg agtggagagg gcggcgggcc ggcgcccctg gccggccgca
     1201 ggaagggagt gagagggcct ggaggccgat aactttgcca tagtctcctc cctccccgga
     1261 actgccccca gcgggtgact ggcagtgtca aggggaattg tcaagacagg acagagaggg
     1321 aagtggtggt ctctgggaga gggtcgggga ggatataagg aatggtgggg gtatcaggga
     1381 caagttgggg ctggggccgg cctgaattcg gtcagattgg gatttgccaa ctatttggaa
     1441 agggcgaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca tctagagggc
     1501 ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta caacgtcgtg
     1561 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     1621 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     1681 atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1741 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1801 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1861 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1921 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1981 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     2041 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     2101 acaaaaattt aacgcgaatt ttaacaaaat tcagggcgca agggctgcta aaggaagcgg
     2161 aacacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg
     2221 gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
     2281 acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     2341 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     2401 ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt
     2461 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     2521 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     2581 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     2641 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     2701 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     2761 caggatctcc tgtcatccca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     2821 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     2881 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     2941 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     3001 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3061 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3121 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3181 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3241 cttgacgagt tcttctgaat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3301 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3361 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3421 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3481 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     3541 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3601 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3661 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3721 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3781 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     3841 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3901 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3961 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     4021 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     4081 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     4141 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4201 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4261 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4321 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4381 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4441 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4501 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4561 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4621 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4681 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4741 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4801 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4861 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4921 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4981 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5041 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     5101 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     5161 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     5221 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     5281 caggaaacag ctatgaccat gattacgcca agcttggtac cgagctcgga tccactagta
     5341 acggccgcca gtgtgctgga attcgccctt ggcaagcttg gagccgacag tgctgagcag
     5401 gcaggagcca agagagggga agcttgagcc tcacgcagtt aggggtgcgc tggagagggt
     5461 ggggcccgac tccgccacac cccaacggtc cttccccctc ctcaccactc ccgcccccac
     5521 ccccaatgga tctgggactg cccctttaag agtagtggcc cctcctccct tcagaggagg
     5581 acctattaga gcctttgccc cggcgtcggt gactcagtgt tcgcgggagc gccgcaccta
     5641 caccagccaa cccagatccc gaggtccgac agcgcccggc ccagatcccc acgcctgcca
     5701 ggagcaagcc gagagccagc cggccggcgc actccgactc cgagcagtct ctgtccttcg
     5761 acccgagc
//
