LOCUS       sequence-394113-        4883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes an mCherry casette under a minimal CMV promoter. Used as the
            target plasmid for plasmid-based integration assays, and for 
            plasmid-based transcriptional activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4883)
  AUTHORS   Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, 
            Tang S, Chavez A, Sternberg SH
  TITLE     Targeted DNA integration in human cells without double-strand breaks
            using CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1.
  PUBMED    36991112
REFERENCE   2  (bases 1 to 4883)
  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"; journalName: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL0341 (pTarget)
FEATURES             Location/Qualifiers
     source          1..4883
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      27..36
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             36..59
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             90..791
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQ
                     TAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFED
                     GGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGE
                     IKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEG
                     RHSTGGMDELYK"
     primer_bind     complement(229..247)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     497..516
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1018..1099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1055..1074)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(1262..1280)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1263..1280)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1298..1314)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1298..1314)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1311..1333)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1322..1338
                     /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)."
     promoter        complement(join(1346..1352,1353..1370,1371..1376))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1391..1412
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1700..2288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1780..1799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2426..2800)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      2797..2806
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3006..3800)
                     /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(3006..3800)
                     /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(3117..3136)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3727..3746
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(4149..4451)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(4251..4270)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     4574..4596
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4588..4605
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4589..4605
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4612..4631
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4612..4630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        4837..4875
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     join(4872..4883,1..13)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 gaaccgtcag atcgcctgga gaattcgcca ccatggacta caaggatgac gacgataaaa
       61 cttccggtgg cggactgggt tccaccgcga gcaagggcga ggaggataac atggccatca
      121 tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg
      181 agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg
      241 tgaccaaggg cggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg
      301 gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc
      361 ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga
      421 cccaggactc ctccctacag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca
      481 acttcccctc cgacggcccc gtaatgcaga agaagacgat gggctgggag gcctcctccg
      541 agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga
      601 aggacggcgg ccactacgac gccgaggtca agaccaccta caaggccaag aagcccgtgc
      661 agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact
      721 acaccatcgt ggaacagtac gagcgcgccg agggccgcca ctccaccggc ggcatggacg
      781 agctgtacaa ggcccgcggt taagatgcat gccagttcta ggaattcgag ctcggtaccc
      841 ggggatcctc tagtcagctg acgcgtgcta gcgcggccgc atcgataagc ttgtcgacga
      901 tatctctaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
      961 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     1021 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1081 aaagcatttt tttcactgcc tcgagcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1141 cggctgcggc gagcggtatc agctcactca aaggcggtaa taaagggcga attccagcac
     1201 actggcggcc gttactagtg gatccgagct cggtaccaag cttgatgcat agcttgagta
     1261 ttctatagtg tcacctaaat agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1321 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1381 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1441 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1501 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1561 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1621 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1681 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1741 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1801 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1861 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1921 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1981 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2041 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2101 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2161 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2221 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2281 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2341 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2401 attaaaaatg aagttttagc acgtgtcagt cctgctcctc ggccacgaag tgcacgcagt
     2461 tgccggccgg gtcgcgcagg gcgaactccc gcccccacgg ctgctcgccg atctcggtca
     2521 tggccggccc ggaggcgtcc cggaagttcg tggacacgac ctccgaccac tcggcgtaca
     2581 gctcgtccag gccgcgcacc cacacccagg ccagggtgtt gtccggcacc acctggtcct
     2641 ggaccgcgct gatgaacagg gtcacgtcgt cccggaccac accggcgaag tcgtcctcca
     2701 cgaagtcccg ggagaacccg agccggtcgg tccagaactc gaccgctccg gcgacgtcgc
     2761 gcgcggtgag caccggaacg gcactggtca acttggccat ggtggccctc ctcacgtgct
     2821 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     2881 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgatgcgg
     2941 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta
     3001 ataattcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg
     3061 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata
     3121 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg
     3181 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
     3241 gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga gcctggcgaa cagttcggct
     3301 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
     3361 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
     3421 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
     3481 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
     3541 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
     3601 agccgcgctg cctcgtcttg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
     3661 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
     3721 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
     3781 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agagcttgat
     3841 cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc
     3901 ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc
     3961 gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt
     4021 gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc
     4081 tgcggactgg ctttctacgt gaaaaggatc taggtgaaga tcctttttga taatctcatg
     4141 cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg
     4201 tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc catatcggtg
     4261 gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggagact
     4321 ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc cggcgtgtca
     4381 ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgtaa
     4441 accttaaact gccgtacgta taggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     4501 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     4561 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac
     4621 tcactatagg gcgaattggg ccctctagat gcatgctcga gcggccgcca gtgtgatgga
     4681 tatctgcaga attcgccctt gcgacgtagg gataacaggg taatcctcag gagactgtgt
     4741 aacaccaatg caggcaaatc aaccttagtc tgaaggccta ggcaaagaag aacacaactt
     4801 acttcttaag gtcccctcca ccccacagtg gggcgaggta ggcgtgtacg gtgggaggcc
     4861 tatataagca gagctcgttt agt
//
