LOCUS       pKG3778_pGEEC585        3256 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 3256)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3256
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     terminator      2..29
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     31..50
                     /label=pENTR_fwd_seq
     primer_bind     31..50
                     /label=oSEQ043 pENTR_fwd_seq
     primer_bind     complement(52..96)
                     /label=oMJ002
     primer_bind     complement(52..92)
                     /label=oMJ010
     primer_bind     complement(52..88)
                     /label=oMJ006
     primer_bind     complement(52..88)
                     /label=oMJ014
     primer_bind     complement(60..88)
                     /label=oMJ019
     primer_bind     93..113
                     /label=oKL204
     promoter        97..283
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(139..156)
                     /label=oSEQ045 phage-dest-rev-2
     primer_bind     complement(263..283)
                     /label=oEP198
     primer_bind     264..283
                     /label=oEP207 EFS-rev
     primer_bind     complement(266..323)
                     /label=oEP200
     misc_feature    284..346
                     /label=hPGK 5' UTR
     primer_bind     307..366
                     /label=oEP199
     primer_bind     347..361
                     /label=oEP197
     regulatory      351..360
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     354..375
                     /label=GG_mRuby2_fwd
     primer_bind     356..375
                     /label=mRuby2_fwd
                     /note="Fwd primer for mRuby2"
     CDS             357..1067
                     /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     357..376
                     /label=oEP159
     primer_bind     complement(383..404)
                     /label=oEP076
     primer_bind     405..424
                     /label=oEP073
     primer_bind     427..446
                     /label=oSEQ062 mRuby2_fwd
     primer_bind     complement(427..446)
                     /label=oSEQ093 mRuby2_rev
     primer_bind     695..714
                     /label=oQT047 mRuby2_1_fwd
     primer_bind     818..837
                     /label=oQT049 mRuby2_2_fwd
     primer_bind     complement(856..875)
                     /label=oQT048 mRuby2_1_rev
     primer_bind     complement(958..977)
                     /label=oQT050 mRuby2_2_rev
     primer_bind     complement(959..978)
                     /label=oSEQ061 mRuby2_rev
     primer_bind     993..1012
                     /label=OSEQ091 mRuby2_fwd_SEQ
     primer_bind     complement(1052..1079)
                     /label=GG_mRuby2_rev
     primer_bind     complement(1052..1074)
                     /label=gemRuby2_rev
     primer_bind     complement(1052..1071)
                     /label=mRuby2_rev
                     /note="Rev primer for mRuby2"
     primer_bind     1070..1094
                     /label=gebGH_fwd
     primer_bind     1076..1097
                     /label=GG_bGH_fwd
     primer_bind     1080..1097
                     /label=GG_WPRE-bGH_fwd
     polyA_signal    1082..1306
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1166..1191)
                     /label=oEP181
                     /note="IVT reverse primer for bGH"
     misc_feature    1189..1191
                     /label=bGH TES
     primer_bind     complement(1288..1310)
                     /label=gebGH_rev
     primer_bind     complement(1289..1305)
                     /label=oSEQ078 bGH_rev
     primer_bind     complement(1292..1306)
                     /label=GG_bGH_rev
     primer_bind     1307..1348
                     /label=oMJ001
     primer_bind     1307..1348
                     /label=oMJ005
     primer_bind     1309..1348
                     /label=oMJ009
     primer_bind     1309..1348
                     /label=oMJ013
     primer_bind     1315..1345
                     /label=oMJ020
     primer_bind     complement(1327..1352)
                     /label=oSEQ033 Pentr_rev
     misc_feature    1329..1352
                     /label=pENTR2B.Seq.REV
     primer_bind     complement(1341..1362)
                     /label=oTA069 mIR-backbone_junction_rev
     primer_bind     complement(1341..1362)
                     /label=oSEQ044 pENTR_rev_seq
     primer_bind     complement(1414..1434)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
     CDS             1428..2237
                     /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(1453..1474)
                     /label=oSEQ023 attl2_rev
     primer_bind     complement(1461..1484)
                     /label=Kan_rev_SEQ
     primer_bind     complement(1851..1880)
                     /label=oTA099 GG_entry_upstream_fwd
     primer_bind     1851..1880
                     /label=oTA102 GG_entry_downstream_rev
     misc_feature    2311..2930
                     /label=pBR322_Origin
     rep_origin      2327..2915
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2359..2378
                     /label=oEP033
     primer_bind     complement(2359..2378)
                     /label=oEP035
     primer_bind     2679..2699
                     /label=oEP024
     primer_bind     complement(2679..2699)
                     /label=oEP025
     primer_bind     2722..2737
                     /label=oTA073 Xho_addition_rev
     primer_bind     complement(2792..2807)
                     /label=oTA072 Xho_addition_for
     primer_bind     2816..2835
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     complement(2932..2951)
                     /label=oEP013
     primer_bind     2932..2951
                     /label=oEP014
     primer_bind     3067..3080
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
     terminator      3080..3166
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3229..3248
                     /label=oSEQ011 pEntr_for
                     /note="25nm	STD"
ORIGIN
        1 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactcttcc tgttagttag
       61 ttacttaagc tcgggcccac ctgcagtcta cttactgggc agagcgcaca tcgcccacag
      121 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      181 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      241 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgctccgga gcgcacgtcg
      301 gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccagaaatg gccaccatgg
      361 tgtctaaggg cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt
      421 cggtcaacgg ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa
      481 ctcaaaccat gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc
      541 ttgccacgtc gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg
      601 atttctttaa acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag
      661 atggtggagt cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc
      721 acgtccaagt cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca
      781 agggttggga gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca
      841 ctcatatggc actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt
      901 acaggtcaaa aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc
      961 gcctggaaag gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag
     1021 ttgccaagtt cgccgggctt ggtggtggga tggacgagct gtacaagtaa caacgttgca
     1081 cctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1141 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1201 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1261 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcgct cgctgactgc
     1321 aggtgcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     1381 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     1441 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     1501 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     1561 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     1621 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     1681 ttatccgtac tcctggtgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     1741 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     1801 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     1861 ttcgtctagc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     1921 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     1981 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     2041 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     2101 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     2161 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     2221 tcgatgagtt tttctaatca gaattggtta attggttgta acattattca gattgggccc
     2281 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2341 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2401 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2461 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2521 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2581 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2641 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2701 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2761 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2821 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2881 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2941 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3001 ctgtggataa ccgtattacc gctagcatgg atctcgggga cgtctaacta ctaagcgaga
     3061 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcggaagact gggcctttcg
     3121 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     3181 tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     3241 caggcatcaa actaag
//
