LOCUS       sequence-273624-        4708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mCherry acceptor plasmid with iCas9 target site upstream of mCherry 
            to be used in plasmid to plasmid integration reporter assay..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4708)
  AUTHORS   Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman 
            DA, Wang X
  TITLE     RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and 
            Integration.
  JOURNAL   CRISPR J. 2019 Aug;2:209-222. doi: 10.1089/crispr.2019.0013.
  PUBMED    31436506
REFERENCE   2  (bases 1 to 4708)
  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: "CRISPR J.";
            date: "2019-08"; volume: "2:209-222. doi"; pages: " 
            10.1089/crispr.2019.0013"
COMMENT     Sequence Label: pUC19-EAMP
FEATURES             Location/Qualifiers
     source          1..4708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    85..106
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(121..126,127..144,145..151)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    159..175
                     /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     164..186
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     183..199
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     183..199
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        291..502
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             515..783
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      911..920
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             917..1624
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1062..1080)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1330..1349
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1631..1684
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1688..2284
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     2181..2201
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(2291..2308)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2291..2307)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2300..2322)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2516..2535)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2635..2657
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2695..2713)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2781..2885
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2886..2954,2955..3746)
                     /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(3104..3123)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3917..4505
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4406..4425
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4659..4676
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
       61 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      121 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      181 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag
      241 aggatccatt aggcggccgc ggatctgcga tcgctccggt gcccgtcagt gggcagagcg
      301 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
      361 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
      421 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
      481 cgggtttgcc gccagaacac agctgaagct tcgaggggct cgcatctctc cttcacgcgc
      541 ccgccgccct acctgaggcc gccatccacg ccggttgagt cgcgttctgc cgcctcccgc
      601 ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc
      661 gggcctttgt ccggcgctcc cttggagcct acctagactc agccggctct ccacgctttg
      721 cctgaccctg cttgctcaac tctacgtctt tgtttcgttt tctgttctgc gccgttacag
      781 atccaagctg tgaccggcgc ctacgctagc ctccgatcct tcccccaggc ttgcactcgt
      841 acgttcgaaa tattataaat tatcagacag accttactcc agaaggggga aggctaggtg
      901 gctaccggtc gccaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
      961 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     1021 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     1081 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     1141 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     1201 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     1261 ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     1321 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     1381 gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg
     1441 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     1501 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     1561 cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta
     1621 caagccccgg gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg
     1681 ccctcgaacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     1741 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     1801 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     1861 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     1921 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     1981 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     2041 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     2101 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     2161 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     2221 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     2281 ctaagaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     2341 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2401 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     2461 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     2521 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     2581 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     2641 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     2701 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2761 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2821 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2881 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2941 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3001 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3061 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3121 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3181 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3241 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3301 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3361 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3421 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3481 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3541 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3601 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3661 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3721 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3781 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3841 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3901 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3961 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4021 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     4081 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4141 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4201 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4261 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4321 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4381 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4441 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4501 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4561 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4621 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4681 cggaagagcg cccaatacgc aaaccgcc
//
