LOCUS       pKG3775_pGEEC582        3494 bp    DNA     circular SYN 23-JAN-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3494)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3494
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     terminator      18..45
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     47..66
                     /label=pENTR_fwd_seq
     primer_bind     47..66
                     /label=oSEQ043 pENTR_fwd_seq
     primer_bind     complement(68..112)
                     /label=oMJ002
     primer_bind     complement(68..108)
                     /label=oMJ010
     primer_bind     complement(68..104)
                     /label=oMJ006
     primer_bind     complement(68..104)
                     /label=oMJ014
     primer_bind     complement(76..104)
                     /label=oMJ019
     primer_bind     109..131
                     /label=oKL206
     promoter        123..571
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     214..233
                     /label=hPGK-mut_fwd
     primer_bind     complement(214..233)
                     /label=hPGK-mut_rev
     primer_bind     complement(552..600)
                     /label=oEP194
     primer_bind     complement(552..571)
                     /label=oEP196
     primer_bind     552..571
                     /label=oEP205 hPGK-rev
     primer_bind     552..570
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     572..620
                     /label=oEP193
     primer_bind     572..615
                     /label=oEP197
     misc_feature    572..600
                     /label=CMV 5' UTR
     primer_bind     599..620
                     /label=oEP195
     regulatory      605..614
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     608..629
                     /label=GG_mRuby2_fwd
     primer_bind     610..629
                     /label=mRuby2_fwd
                     /note="Fwd primer for mRuby2"
     CDS             611..1321
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     primer_bind     611..630
                     /label=oEP159
     primer_bind     complement(637..658)
                     /label=oEP076
     primer_bind     659..678
                     /label=oEP073
     primer_bind     681..700
                     /label=oSEQ062 mRuby2_fwd
     primer_bind     complement(681..700)
                     /label=oSEQ093 mRuby2_rev
     primer_bind     complement(1213..1232)
                     /label=oSEQ061 mRuby2_rev
     primer_bind     1247..1266
                     /label=OSEQ091 mRuby2_fwd_SEQ
     primer_bind     complement(1306..1333)
                     /label=GG_mRuby2_rev
     primer_bind     complement(1306..1328)
                     /label=gemRuby2_rev
     primer_bind     complement(1306..1325)
                     /label=mRuby2_rev
                     /note="Rev primer for mRuby2"
     primer_bind     1324..1348
                     /label=gebGH_fwd
     primer_bind     1330..1351
                     /label=GG_bGH_fwd
     primer_bind     1334..1351
                     /label=GG_WPRE-bGH_fwd
     polyA_signal    1336..1560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1420..1445)
                     /label=oEP181 IVT-bGH-rev
     misc_feature    1443..1445
                     /label=bGH TES
     primer_bind     complement(1542..1564)
                     /label=gebGH_rev
     primer_bind     complement(1543..1559)
                     /label=oSEQ078 bGH_rev
     primer_bind     complement(1546..1560)
                     /label=GG_bGH_rev
     primer_bind     1561..1602
                     /label=oMJ001
     primer_bind     1561..1602
                     /label=oMJ005
     primer_bind     1563..1602
                     /label=oMJ009
     primer_bind     1563..1602
                     /label=oMJ013
     primer_bind     1569..1599
                     /label=oMJ020
     primer_bind     complement(1581..1606)
                     /label=oSEQ033 Pentr_rev
     misc_feature    1583..1606
                     /label=pENTR2B.Seq.REV
     primer_bind     complement(1595..1616)
                     /label=oTA069 mIR-backbone_junction_rev
     primer_bind     complement(1595..1616)
                     /label=oSEQ044 pENTR_rev_seq
     primer_bind     complement(1668..1688)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
     CDS             1682..2491
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1707..1728)
                     /label=oSEQ023 attl2_rev
     primer_bind     complement(1715..1738)
                     /label=Kan_rev_SEQ
     primer_bind     complement(2105..2134)
                     /label=oTA099 GG_entry_upstream_fwd
     primer_bind     2105..2134
                     /label=oTA102 GG_entry_downstream_rev
     misc_feature    2565..3184
                     /label=pBR322_Origin
     rep_origin      2581..3169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2613..2632
                     /label=oEP033
     primer_bind     complement(2613..2632)
                     /label=oEP035
     primer_bind     2933..2953
                     /label=oEP024
     primer_bind     complement(2933..2953)
                     /label=oEP025
     primer_bind     2976..2991
                     /label=oTA073 Xho_addition_rev
     primer_bind     complement(3046..3061)
                     /label=oTA072 Xho_addition_for
     primer_bind     3070..3089
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     complement(3186..3205)
                     /label=oEP013
     primer_bind     3186..3205
                     /label=oEP014
     primer_bind     3321..3334
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      3334..3420
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(3483..3494,1..8)
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
ORIGIN
        1 caggcatcaa actaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
       61 tcttcctgtt agttagttac ttaagctcgg gcccacctgc agtctactta ctataaggat
      121 ccggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
      181 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg gatctcgcac attcttcacg
      241 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
      301 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
      361 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca
      421 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
      481 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
      541 ctgcccgcgc ggtgttccgc attctgcaag cagatcgcct ggagacgcca tccacgctgt
      601 aatggccacc atggtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt
      661 ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa
      721 tccgtacatg ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt
      781 tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc
      841 gaaaggcatt cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt
      901 tacgagatac gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg
      961 ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat
     1021 gcagaagaag accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg
     1081 tctgagggga tacactcata tggcactgaa agttgatggt ggtggccatc tgtcttgctc
     1141 tttcgtaaca acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtatcca
     1201 tgccgttgat caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca
     1261 acgcgaacac gcagttgcca agttcgccgg gcttggtggt gggatggacg agctgtacaa
     1321 gtaacaacgt tgcacctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1381 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1441 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1501 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     1561 cgctcgctga ctgcaggtgc ccgtgtctca aaatctctga tgttacattg cacaagataa
     1621 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     1681 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     1741 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     1801 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     1861 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     1921 gaccatcaag cattttatcc gtactcctgg tgatgcatgg ttactcacca ctgcgatccc
     1981 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2041 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2101 cagcgatcgc gtatttcgtc tagctcaggc gcaatcacga atgaataacg gtttggttga
     2161 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2221 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2281 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     2341 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     2401 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     2461 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacatta
     2521 ttcagattgg gccccgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2581 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2641 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2701 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2761 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2821 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2881 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2941 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3001 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3061 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3121 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3181 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3241 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta
     3301 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
     3361 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3421 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     3481 ccgccataaa ctgc
//
