LOCUS       sequence-333252-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For creating a stable mCherry-GAL9 cell line.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Munson MJ, O'Driscoll G, Silva AM, Lazaro-Ibanez E, Gallud A, Wilson
            JT, Collen A, Esbjorner EK, Sabirsh A
  TITLE     A high-throughput Galectin-9 imaging assay for quantifying 
            nanoparticle uptake, endosomal escape and functional RNA delivery.
  JOURNAL   Commun Biol. 2021 Feb 16;4(1):211. doi: 10.1038/s42003-021-01728-8.
  PUBMED    33594247
REFERENCE   2  (bases 1 to 6671)
  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: "Commun 
            Biol."; date: "2021-02-16"; volume: "4(1):211. doi"; pages: " 
            10.1038/s42003-021-01728-8"
COMMENT     Sequence Label: mCherry-GAL9
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     254..276
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     268..285
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     269..285
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     296..315
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        296..314
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    402..427
                     /label=SA
                     /note="splice acceptor site"
     CDS             451..504
                     /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             514..1113
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(514..533)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1010..1030
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1267..1378
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1590..1606)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1603..1625)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1614..1630
                     /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(1638..1644,1645..1662,1663..1668))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1683..1704
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1967..2178
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             2191..2459
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      2500..2509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2506..3213
                     /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(2651..2669)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2919..2938
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4326..4550
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     terminator      4757..4800
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4857..5445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4937..4956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5616..6407,6408..6476))
                     /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     6239..6258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6477..6540)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
       61 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg gccgctacag
      121 ggcgctccca ttcgccattc aggctgcgca actgttggga agggcgtttc ggtgcgggcc
      181 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      241 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgacgtaata
      301 cgactcacta tagggcgaat tgtaggcgga aggccgtcaa ggcctaggcg cgccagcatc
      361 tagatacgcg tatcctgtcc ctaggccacc cactgtgggg tctgacctct tctcttcctc
      421 ccacagggcc tcgagagatc tggcagcgga gagggcagag gaagtcttct aacatgcggt
      481 gacgtggagg agaatcccgg ccctaggctc gaaatgaccg agtacaagcc cacggtgcgc
      541 ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac
      601 taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg
      661 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
      721 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
      781 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
      841 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
      901 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
      961 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     1021 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1081 acctggtgca tgacccgcaa gcccggtgcc taacgcccgc cccacgaccc gcagcgcccg
     1141 accgaaagga gcgcacgacc ccatggctcc gaccgaagcc acccggggcg gccccgccga
     1201 ccccgcaccc gcccccgagg cccaccgact ctagagggcc cgtttaaacc cgctgatcag
     1261 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1321 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1381 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1441 aggattggga agacaatagc aaccggtgct agaattctca tgatatcttc cgtcgacgct
     1501 ctctagagct agcggccgct actagacgcg tgatcggatc ccgggcccgt cgactgcaga
     1561 ggcctgcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     1621 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     1681 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     1741 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1801 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1861 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1921 gcaggaaaga acatgtggat ctgcgatcgc tccggtgccc gtcagtgggc agagcgcaca
     1981 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga
     2041 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag
     2101 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg
     2161 tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc acgcgcccgc
     2221 cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt
     2281 ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc
     2341 ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg
     2401 accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg ttacagatcc
     2461 aagctgtgac cggcgcctac actagtccag tgtggtggag ccaccatggt gagcaagggc
     2521 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     2581 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     2641 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     2701 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     2761 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     2821 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     2881 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
     2941 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     3001 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     3061 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     3121 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     3181 cactccaccg gcggcatgga cgagctgtac aagatcggag gaggaggatc cggaggagga
     3241 ggatccggag gaggaggatc catggccttc agcggttccc aggctcccta cctgagtcca
     3301 gctgtcccct tttctgggac tattcaagga ggtctccagg acggacttca gatcactgtc
     3361 aatgggaccg ttctcagctc cagtggaacc aggtttgctg tgaactttca gactggcttc
     3421 agtggaaatg acattgcctt ccacttcaac cctcggtttg aagatggagg gtacgtggtg
     3481 tgcaacacga ggcagaacgg aagctggggg cccgaggaga ggaagacaca catgcctttc
     3541 cagaagggga tgccctttga cctctgcttc ctggtgcaga gctcagattt caaggtgatg
     3601 gtgaacggga tcctcttcgt gcagtacttc caccgcgtgc ccttccaccg tgtggacacc
     3661 atctccgtca atggctctgt gcagctgtcc tacatcagct tccagaaccc ccgcacagtc
     3721 cctgttcagc ctgccttctc cacggtgccg ttctcccagc ctgtctgttt cccacccagg
     3781 cccagggggc gcagacaaaa aacccagaca gtcatccaca cagtgcagag cgcccctgga
     3841 cagatgttct ctactcccgc catcccacct atgatgtacc cccaccccgc ctatccgatg
     3901 cctttcatca ccaccattct gggagggctg tacccatcca agtccatcct cctgtcaggc
     3961 actgtcctgc ccagtgctca gaggttccac atcaacctgt gctctgggaa ccacatcgcc
     4021 ttccacctga acccccgttt tgatgagaat gctgtggtcc gcaacaccca gatcgacaac
     4081 tcctgggggt ctgaggagcg aagtctgccc cgaaaaatgc ccttcgtccg tggccagagc
     4141 ttctcagtgt ggatcttgtg tgaagctcac tgcctcaagg tggccgtgga tggtcagcac
     4201 ctgtttgaat actaccatcg cctgaggaac ctgcccacca tcaacagact ggaagtgggg
     4261 ggcgacatcc agctgaccca tgtgcagaca tagagggccc gtttaaaccc gctgatcagc
     4321 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4381 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4441 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4501 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg acacatgtga
     4561 gcaaaaggcc agcaaatttt aattaactgg cctcatgggc cttccgccta tcactgcccg
     4621 ctttccagtc gggaaacctg tcgtgccagc tgcattaaca tggtcatagc tgtttccttg
     4681 cgtattgggc gctctccgct tcctcgctca ctgactcgct gcgctcggtc gttcgggtaa
     4741 agcctggggt gcctaaataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
     4801 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4861 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4921 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4981 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5041 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5101 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5161 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5221 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5281 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5341 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5401 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5461 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5521 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5581 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5641 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5701 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagaa
     5761 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5821 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5881 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5941 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6001 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6061 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6121 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6181 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6241 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6301 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6361 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6421 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6481 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6541 tttgaagcta ataacaggcc tgctggtaat cgcaggcctt tttatttggt gtatttagaa
     6601 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     6661 gttaatattt t
//
