LOCUS       pKG3774_pGEEC580        3246 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 3246)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3246
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     terminator      10..37
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     39..58
                     /label=pENTR_fwd_seq
     primer_bind     39..58
                     /label=oSEQ043 pENTR_fwd_seq
     primer_bind     complement(60..104)
                     /label=oMJ002
     primer_bind     complement(60..100)
                     /label=oMJ010
     primer_bind     complement(60..96)
                     /label=oMJ006
     primer_bind     complement(60..96)
                     /label=oMJ014
     primer_bind     complement(68..96)
                     /label=oMJ019
     promoter        105..343
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(165..182)
                     /label=oSEQ045 phage-dest-rev-2
     primer_bind     290..309
                     /label=oEP207 EFS-rev
     primer_bind     309..323
                     /label=oEP182 IVT-EFS-fwd
     misc_feature    310..315
                     /label=EF1a TSS
     primer_bind     313..332
                     /label=oEP209 EF1a-rev
     primer_bind     315..350
                     /label=oEP189
     primer_bind     complement(318..350)
                     /label=oEP190
     misc_feature    333..336
                     /label=INTRON REMOVAL SITE
     regulatory      349..358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     352..373
                     /label=GG_mRuby2_fwd
     primer_bind     354..373
                     /label=mRuby2_fwd
                     /note="Fwd primer for mRuby2"
     CDS             355..1065
                     /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     355..374
                     /label=oEP159
     primer_bind     complement(381..402)
                     /label=oEP076
     primer_bind     403..422
                     /label=oEP073
     primer_bind     425..444
                     /label=oSEQ062 mRuby2_fwd
     primer_bind     complement(425..444)
                     /label=oSEQ093 mRuby2_rev
     primer_bind     complement(957..976)
                     /label=oSEQ061 mRuby2_rev
     primer_bind     991..1010
                     /label=OSEQ091 mRuby2_fwd_SEQ
     primer_bind     complement(1050..1077)
                     /label=GG_mRuby2_rev
     primer_bind     complement(1050..1072)
                     /label=gemRuby2_rev
     primer_bind     complement(1050..1069)
                     /label=mRuby2_rev
                     /note="Rev primer for mRuby2"
     primer_bind     1068..1092
                     /label=gebGH_fwd
     primer_bind     1074..1095
                     /label=GG_bGH_fwd
     primer_bind     1078..1095
                     /label=GG_WPRE-bGH_fwd
     polyA_signal    1080..1304
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1164..1189)
                     /label=oEP181 IVT-bGH-rev
     misc_feature    1187..1189
                     /label=bGH TES
     primer_bind     complement(1286..1308)
                     /label=gebGH_rev
     primer_bind     complement(1287..1303)
                     /label=oSEQ078 bGH_rev
     primer_bind     complement(1290..1304)
                     /label=GG_bGH_rev
     primer_bind     1305..1346
                     /label=oMJ001
     primer_bind     1305..1346
                     /label=oMJ005
     primer_bind     1307..1346
                     /label=oMJ009
     primer_bind     1307..1346
                     /label=oMJ013
     primer_bind     1313..1343
                     /label=oMJ020
     primer_bind     complement(1325..1350)
                     /label=oSEQ033 Pentr_rev
     misc_feature    1327..1350
                     /label=pENTR2B.Seq.REV
     primer_bind     complement(1339..1360)
                     /label=oTA069 mIR-backbone_junction_rev
     primer_bind     complement(1339..1360)
                     /label=oSEQ044 pENTR_rev_seq
     primer_bind     complement(1412..1432)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
     CDS             1426..2235
                     /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(1451..1472)
                     /label=oSEQ023 attl2_rev
     primer_bind     complement(1459..1482)
                     /label=Kan_rev_SEQ
     primer_bind     complement(1849..1878)
                     /label=oTA099 GG_entry_upstream_fwd
     primer_bind     1849..1878
                     /label=oTA102 GG_entry_downstream_rev
     misc_feature    2309..2928
                     /label=pBR322_Origin
     rep_origin      2325..2913
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2357..2376
                     /label=oEP033
     primer_bind     complement(2357..2376)
                     /label=oEP035
     primer_bind     2677..2697
                     /label=oEP024
     primer_bind     complement(2677..2697)
                     /label=oEP025
     primer_bind     2720..2735
                     /label=oTA073 Xho_addition_rev
     primer_bind     complement(2790..2805)
                     /label=oTA072 Xho_addition_for
     primer_bind     2814..2833
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     complement(2930..2949)
                     /label=oEP013
     primer_bind     2930..2949
                     /label=oEP014
     primer_bind     3065..3078
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      3078..3164
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3227..3246
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
ORIGIN
        1 aaactaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg
       61 ttagttagtt acttaagctc gggcccacct gcagtctact tactgctccg gtgcccgtca
      121 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
      181 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
      241 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
      301 tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgagaatggc caccatggtg
      361 tctaagggcg aagagctgat caaggaaaat atgcgtatga aggtggtcat ggaaggttcg
      421 gtcaacggcc accaattcaa atgcacaggt gaaggagaag gcaatccgta catgggaact
      481 caaaccatga ggatcaaagt catcgaggga ggacccctgc catttgcctt tgacattctt
      541 gccacgtcgt tcatgtatgg cagccgtact tttatcaagt acccgaaagg cattcctgat
      601 ttctttaaac agtcctttcc tgagggtttt acttgggaaa gagttacgag atacgaagat
      661 ggtggagtcg tcaccgtcat gcaggacacc agccttgagg atggctgtct cgtttaccac
      721 gtccaagtca gaggggtaaa ctttccctcc aatggtcccg tgatgcagaa gaagaccaag
      781 ggttgggagc ctaatacaga gatgatgtat ccagcagatg gtggtctgag gggatacact
      841 catatggcac tgaaagttga tggtggtggc catctgtctt gctctttcgt aacaacttac
      901 aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta tccatgccgt tgatcaccgc
      961 ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag tacaacgcga acacgcagtt
     1021 gccaagttcg ccgggcttgg tggtgggatg gacgagctgt acaagtaaca acgttgcacc
     1081 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1141 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1201 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1261 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcgctcg ctgactgcag
     1321 gtgcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     1381 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     1441 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     1501 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     1561 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     1621 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     1681 atccgtactc ctggtgatgc atggttactc accactgcga tccccggaaa aacagcattc
     1741 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     1801 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     1861 cgtctagctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     1921 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     1981 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     2041 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     2101 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     2161 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     2221 gatgagtttt tctaatcaga attggttaat tggttgtaac attattcaga ttgggccccg
     2281 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2341 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2401 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2461 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2521 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2581 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2641 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2701 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2761 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2821 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2881 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2941 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3001 gtggataacc gtattaccgc tagcatggat ctcggggacg tctaactact aagcgagagt
     3061 agggaactgc caggcatcaa ataaaacgaa aggctcagtc ggaagactgg gcctttcgtt
     3121 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     3181 tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     3241 ggcatc
//
