LOCUS       sequence-346362-        7929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCherry mRNA from the Drosophila Metallothionein A 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7929)
  AUTHORS   Ai Y, Liang D, Wilusz JE
  TITLE     CRISPR/Cas13 effectors have differing extents of off-target effects 
            that limit their utility in eukaryotic cells.
  JOURNAL   Nucleic Acids Res. 2022 Mar 4. pii: 6542487. doi: 
            10.1093/nar/gkac159.
  PUBMED    35244715
REFERENCE   2  (bases 1 to 7929)
  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: "Nucleic 
            Acids Res. 2022 Mar 4. pii: 6542487. doi: 10.1093/nar/gkac159."
COMMENT     Sequence Label: Hy_pMtnA mCherry SV40
FEATURES             Location/Qualifiers
     source          1..7929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(43..631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(123..142)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(802..1593,1594..1662))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1425..1444
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1663..1767)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1835..1853
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3249..3528
                     /label=copia promoter
                     /note="strong promoter from the Drosophila transposable 
                     element copia (Sinclair et al., 1986)"
     CDS             3574..4599
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3574..4599
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     intron          4962..5027
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5157..5177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5602..5736
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        5737..6163
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     CDS             6182..6247
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6254..6961
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(6396..6414)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     6664..6683
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    6970..7091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7801..7818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
       61 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
      421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
      481 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
      541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
      601 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
      661 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
      721 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      781 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      841 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      901 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
      961 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1021 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1081 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     1141 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1201 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1261 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1321 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1381 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     1441 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     1501 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     1561 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     1621 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1681 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1741 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     1801 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     1861 tcttcaagaa ttcacatttg tacgaatttt ttttttatca aaagttcgag tttttcacca
     1921 atttcctcat caaccgagca aggcaaacgg ctttgaataa tatggtgtta tatatacata
     1981 tatcaaatcg ctgctgactg cgtgattgat ggccccaaga ttacatatta tcgaatcagg
     2041 attcagaagg agatcaatgt caaatgcgga caggaacatg aaagacgcct gttatgcgca
     2101 attaaaaatt tgggtttaat tgctgtggaa actgttgttg gcggcatctt aagttcctgt
     2161 ttaacaacat caactactta tgtacgtaga agcgtttaag ccatttgcat acagatgaga
     2221 actggctttt gtgctaatca gtcaagatga ctccgatgat gatgactcat tacctgacca
     2281 gttttcgctg ctttcttttc aacaactact tgtatatgta ttgtatccaa tagcaataca
     2341 ttgaatttcc atggtctagt cacgtattat catttaattg acaccaagtc gtgttattgt
     2401 tgagctatcg agttcagctc aaacatttct tattcccatg aataagccgg caaaaatatg
     2461 caatctatga aagttaatat aagcaaacct tactttgact caataccaat gcactttgtg
     2521 tcgataggtt cacgcaattg aggcgattat tccgataacc caagcgattg actgttcccg
     2581 tttcgattcc aattgaaatt tggaaatgta caatagtttt gctatatgct gtcaagtacg
     2641 ctcttatctt ctctgggttt tcttcagagt ttcgaaacgc ttcttctttt ttttgttttt
     2701 ttttttttgg aatctcgtat tttggaaggg gctcccctct ggaatttgtt acactgtcgt
     2761 tatcattgcg aacaagcggc ccgaagctat cagcgacttt aacatttaca atgcactttt
     2821 ttacgaccaa ttaaatgtac attttccttt cttcgcccgt tgataagcga acgcgatgtg
     2881 gcgcaggcaa tgtgttgctc ttgcgacaca aacgcaatca aaatggattc aatttcgctt
     2941 tttcccagtg aaacgaagaa cgaaccgacc atcatgatat gctcctctgc atgttgcgta
     3001 ttgaatcaat gacaatttca attaagccgc ccgttcgtca tgcgttttcg tgcgcttcga
     3061 aatgctgata acgctgctgt cctccaactg ctttgcatgt ggacacaatt ccatttattt
     3121 aattctttta tttggatcgg ttaaattaaa aagcgccttg ttacgcattt aacgttgttt
     3181 ccggtgcgtg gtggtttcat gcttctggga acggcaaatg ggtttaggat tgggaacccc
     3241 tcatcatctg ttggaatata ctattcaacc tacaaaagta acgttaaaca acactacttt
     3301 atatttgata tgaatggcca caccttttat gccataaaac atattgtaag agaataccac
     3361 tctttttatt ccttctttcc ttcttgtacg ttttttgctg taagtaggtc gtggtgctgg
     3421 tgttgcagtt gaaataactt aaaatataaa tcataaaact caaacataaa cttgactatt
     3481 tatttattta ttaagaaagg aaatataaat tataaattac aacaggttat ggacctgcag
     3541 ccaagcttgg cgctcgtccg ggggcaatga gatatgaaaa agcctgaact caccgcgacg
     3601 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3661 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3721 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3781 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3841 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3901 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3961 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     4021 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     4081 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     4141 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     4201 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     4261 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     4321 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     4381 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     4441 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4501 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4561 aaccgacgcc ccagcactcg tccgagggca aaggaataga gtagatgccg accgaacaag
     4621 agctgatttc gagaacgcct cagccagcaa ctcgcgcgag cctagcaagg caaatgcgag
     4681 agaacggcct tacgcttggt ggcacagttc tcgtccacag ttcgctaagc tcgctcggct
     4741 gggtcgcggg agggccggtc gcagtgattc aggcccttct ggattgtgtt ggtccccagg
     4801 gcacgattgt catgcccacg cactcgggtg atctgactga tcccgcagat tggagatcgc
     4861 cgcccgtgcc tgccgattgg gtgcagatct ttgtgaagga accttacttc tgtggtgtga
     4921 cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa
     4981 gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg
     5041 gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag
     5101 aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa
     5161 aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga
     5221 gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa
     5281 aagctgcact gctatacaag aaaattatgg aaaaatattc tgtaaccttt ataagtaggc
     5341 ataacagtta taatcataac atactgtttt ttcttactcc acacaggcat agagtgtctg
     5401 ctattaataa ctatgctcaa aaattgtgta cctttagctt tttaatttgt aaaggggtta
     5461 ataaggaata tttgatgtat agtgccttga ctagagatca taatcagcca taccacattt
     5521 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     5581 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     5641 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5701 tccaaactca tcaatgtatc ttatcatgtc tggatcaatt cgttgcagga caggatgtgg
     5761 tgcccgatgt gactagctct ttgctgcagg ccgtcctatc ctctggttcc gataagagac
     5821 ccagaactcc ggccccccac cgcccaccgc cacccccata catatgtggt acgcaagtaa
     5881 gagtgcctgc gcatgcccca tgtgccccac caagagcttt gcatcccata caagtcccca
     5941 aagtggagaa ccgaaccaat tcttcgcggg cagaacaaaa gcttctgcac acgtctccac
     6001 tcgaatttgg agccggccgg cgtgtgcaaa agaggtgaat cgaacgaaag acccgtgtgt
     6061 aaagccgcgt ttccaaaatg tataaaaccg agagcatctg gccaatgtgc atcagttgtg
     6121 gtcagcagca aaatcaagtg aatcatctca gtgcaactaa aggggggatc tcgagaccat
     6181 ggactacaaa gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga
     6241 tgacaagacc ggtgtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     6301 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     6361 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     6421 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     6481 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     6541 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     6601 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     6661 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     6721 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     6781 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     6841 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
     6901 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta
     6961 ggcggccgca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     7021 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     7081 aatgtatctt aactagtgga tccactaggg gccgccgacg cgaggctgga tggccttccc
     7141 cattatgatt cttctcgctt ccggcggcat cgggatgccc gcgttgcagg ccatgctgtc
     7201 caggcaggta gatgacgacc atcagggaca gcttcaagga tcgctcgcgg ctcttaccag
     7261 cctaacttcg atcattggac cgctgatcgt cacggcgatt tatgccgcct cggcgagcac
     7321 atggaacggg ttggcatgga ttgtaggcgc cgccctatac cttgtctgcc tccccgcgtt
     7381 gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg gcacctcgct
     7441 aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac tgtgaatgcg
     7501 caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag ccgcacgcgg
     7561 cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg
     7621 aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc
     7681 gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg
     7741 gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgct cttccgcttc
     7801 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     7861 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     7921 aaaaggcca
//
