LOCUS       sequence-482033-        6247 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6247)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 6247)
  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"
COMMENT     Sequence Label: pMC-dest-BsaI-cmlc2:mCherry
FEATURES             Location/Qualifiers
     source          1..6247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(261..278)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    299..332
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     polyA_signal    456..590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(493..512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     547..566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(620..1330)
                     /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(898..917)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1167..1185
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3075..3869)
                     /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(3075..3869)
                     /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(3186..3205)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3796..3815
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     terminator      4118..4204
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4296..4323
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4457..5045
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4946..4965
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5199..5216
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5231..5371
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5297..5316)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5457..5479
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acattaccct gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta
       61 tcccagatga cattaccctg ttatccctag atacattacc ctgttatccc agatgacata
      121 ccctgttatc cctagatgac attaccctgt tatcccagat gacattaccc tgttatccct
      181 agatacatta ccctgttatc ccagatgaca ttaccctgtt atccctagat gacattaccc
      241 tgttatccca gataaactca atgatgatga tgatgatggt cgagactcag cggccgcggt
      301 gccagggcgt gcccttgggc tccccgggcg cgactagtga attccagtag agaccatcga
      361 tgctagcgag ctcggtctct ccaagtcgac aattaaaaaa cctcccacac ctccccctga
      421 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
      481 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
      541 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tacgtaatac
      601 gactcactat agttctagac tacttgtaca gctcgtccat gccgccggtg gagtggcggc
      661 cctcggcgcg ttcgtactgt tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca
      721 acttgatgtt gacgttgtag gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg
      781 tcttgacctc agcgtcgtag tggccgccgt ccttcagctt cagcctctgc ttgatctcgc
      841 ccttcagggc gccgtcctcg gggtacatcc gctcggagga ggcctcccag cccatggtct
      901 tcttctgcat tacggggccg tcggagggga agttggtgcc gcgcagcttc accttgtaga
      961 tgaactcgcc gtcctgcagg gaggagtcct gggtcacggt caccacgccg ccgtcctcga
     1021 agttcatcac gcgctcccac ttgaagccct cggggaagga cagcttcaag tagtcgggga
     1081 tgtcggctgg gtgcttcacg taggccttgg agccgtacat gaactgaggg gacaggatgt
     1141 cccaggcgaa gggcaggggg ccacccttgg tcaccttcag cttggcggtc tgggtgccct
     1201 cgtaggggcg gccctcgccc tcgccctcga tctcgaactc gtggccgttc acggagccct
     1261 ccatgtgcac cttgaagcgc atgaactcct tgatgatggc catgttatcc tcctcgccct
     1321 tgctcaccat gaattcggag aagacattgg aagagcctct ttgagcagtt ttccccctct
     1381 tggccgcggc ttttttacta gcctgtgagt aaaaaaaaag agaagcctta tttggaaact
     1441 caatttatag catttataca ctgtaaaacc caatagtcaa ctttatcaaa tgaaatgagt
     1501 atagttaact caaaattggc tgaaaattaa ttctacttat ttgaaaagag ttttgaactt
     1561 agtgttgagg gtaatgagtt aattaattac ctcattactt caacttaaat ggagtaagtt
     1621 cacagtactc atatagattc ttttttttaa ctcaaatggt ttgtagcaat cggtttcctc
     1681 taacggtgtg agctgcctca acttattggg ttttacagta ctcagttggt tgagctctct
     1741 tcatttactg ggttttactg tgctcaaatt gcttctttta ctcaaataga ttaagttcac
     1801 agtactcatt tagattagtt tttgaacttc agatggcttg ttacaatcgg ttttctcaaa
     1861 tgatttgagt taccttaact tttttgcgtt ttacagtgta cagaaaaaag tacctaagta
     1921 aaaatactca ttaaaagata gaatcacaac atacaaatgc agaaaatgat gcttctaata
     1981 tttaatatca acaaacaaac accatttaac ttcatataga cccttttcat tttcacatta
     2041 atattatatt caaaaccaaa aaagtatagc tttgtctact caccatgttc actgtctgct
     2101 ttgctgttgg tctgggctcc tgggtcactg acgtttctaa tggagtcttt atgtatgagg
     2161 actcttatca tttgttcttc tataaaggtc tgcagtgttt ctgttcgtcc cctacatgga
     2221 cacccagagc ctcctaaata caggagccct gataactgca caagtgcaca gattccagca
     2281 gggtggaaaa tgagataaag tgtgcagatg gggaggggga cgtgaatgag agatttgagg
     2341 gatgaaaagg atggatgaat gcattgaaaa tagccccttt cacaaaataa taccagtaaa
     2401 ttgtcatata attaatgacc agacctttac ctgtaaatga tgtgatgttc atatacagaa
     2461 gaatgccaac aatttacaga tgattttaca aattcttaat tttccttttc taaatggatt
     2521 tgttcacaca ttatcttttc aaagcaattg aattttctgg aaaagattgg ctgtgtgtgt
     2581 aaaaggtgct actgaggaaa ttaatgtgac atggaatgaa gcaaacagca gcctaagaca
     2641 ggatggcagg aaaaatgtca catgtttaaa ataagagtga aaccaacgac ctggacttat
     2701 agagactgtg tgtccttagg caaagctctt gtacaaacag tgcttgtgac tgtgaaattc
     2761 ggggtttgcc tggattgtgt taaatgtgtg cttggatgta tcataaaaaa gaatcggttc
     2821 tctgtatttg acattttctg taatttatct aaagttacaa ctggctatgc ctgattttta
     2881 tcactattga atcaatcatt ttaaagaatg ttaagtctgc tgatttggag agctcattta
     2941 cagggcccgc cccaactggg gtaacctttg agttctctca gttgggggta atcagcatca
     3001 tgatgtggta ccacatcatg atgctgatta taagaatgcg gccgccacac tctagtggat
     3061 ctcgagttaa taattcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     3121 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     3181 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     3241 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     3301 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc tcgccttgag cctggcgaac
     3361 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     3421 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     3481 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     3541 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     3601 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     3661 agccacgata gccgcgctgc ctcgtcttgc agttcattca gggcaccgga caggtcggtc
     3721 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     3781 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     3841 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     3901 gagcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     3961 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
     4021 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc
     4081 tttgctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     4141 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     4201 aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
     4261 acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct
     4321 ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc
     4381 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4441 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4501 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4561 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4621 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4681 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4741 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4801 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4861 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4921 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4981 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5041 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5101 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5161 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5221 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     5281 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5341 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5401 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5461 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa
     5521 ttcgcgcgcg aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat
     5581 tctgcaaacc ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga
     5641 caacttgacg gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct
     5701 ggccccggtg cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt
     5761 gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat
     5821 acgttggtcc tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga
     5881 cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg gcgattcttc tctcatccgc
     5941 caaaacagcc aagctgtaga ccgtttgaca ttaccctgtt atccctagat acattaccct
     6001 gttatcccag atgacatacc ctgttatccc tagatgacat taccctgtta tcccagatga
     6061 cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt accctgttat
     6121 ccctagatac attaccctgt tatcccagat gacataccct gttatcccta gatgacatta
     6181 ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct gttatcccag
     6241 atgacat
//
