LOCUS       Exported                4657 bp ds-DNA     circular SYN 27-FEB-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4657)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Feb 28, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4657
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(47..66)
                     /label=oSEQ043 pENTR_fwd_seq
                     /note="color: black; sequence: gcgtttctacaaactcttcc; added:
                     2025-02-27"
     terminator      68..95
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
                     /note="color: #ffffff"
     primer_bind     complement(105..124)
                     /label=oSEQ011 pEntr_for
                     /note="color: black; sequence: GCCATAAACTGCCAGGCATC; added:
                     2025-02-27"
     terminator      187..273
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
                     /note="color: #ffffff"
     rep_origin      complement(438..1026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     complement(518..537)
                     /label=oSEQ067 pBR322-ori_fwd
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT; added:
                     2025-02-27"
     CDS             complement(1116..1925)
                     /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"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1869..1892
                     /label=OSEQ087 Kan_rev_SEQ
                     /note="color: black; sequence: AGCATCCATGTTGGAATTTAATCG;
                     added: 2025-02-27"
     primer_bind     1879..1900
                     /label=oSEQ023 attl2_rev
                     /note="color: black; sequence: TTGGAATTTAATCGCGGCCTCG;
                     added: 2025-02-27"
     primer_bind     1919..1939
                     /label=oSEQ005 pENTR_rev
                     /note="color: black; sequence: ggctcataacaccccttgtat;
                     added: 2025-02-27"
     primer_bind     1991..2012
                     /label=oSEQ044 pENTR_rev_seq
                     /note="color: black; sequence: cttgtgcaatgtaacatcagag;
                     added: 2025-02-27"
     primer_bind     2001..2026
                     /label=?oSEQ033 Pentr_rev
                     /note="color: black; sequence: GTAACATCAGAGATTTTGAGACACGG; 
                     added: 2025-02-27"
     primer_bind     2005..2024
                     /label=GW_ATTL2
                     /note="color: black; sequence: CATCAGAGATTTTGAGACAC; added:
                     2025-02-27"
     promoter        2047..3228
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    2065..2276
                     /label=EF-1a core promoter
                     /note="color: #ffffff"
     primer_bind     complement(2107..2124)
                     /label=oSEQ045 phage-dest-rev-2
                     /note="color: black; sequence: TTCAATTGCCGACCCCTC; added: 
                     2025-02-27"
     intron          2277..3219
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human
                     EF-1-alpha"
                     /note="color: #------"
     primer_bind     complement(3015..3036)
                     /label=oSEQ046 phage-dest-upstream-seq
                     /note="color: black; sequence: CGACGTACTCCAAAAGCTCGAG;
                     added: 2025-02-27"
     primer_bind     3079..3098
                     /label=oSEQ064 EF1a_fwd
                     /note="color: black; sequence: CACACTGAGTGGGTGGAGAC; added:
                     2025-02-27"
     regulatory      3283..3292
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             3289..3336
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /note="color: #ff0000"
                     /translation="MVSKGEELIKENMRMK"
     ncRNA           3375..3467
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /note="color: #8fbc8f; direction: RIGHT"
                     /ncRNA_class="miRNA"
     misc_feature    3468..3570
                     /label=FF4 Hairpin
                     /note="color: #a6acb3"
     ncRNA           3571..3685
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /note="color: #8fbc8f; direction: RIGHT"
                     /ncRNA_class="miRNA"
     CDS             3714..4376
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /note="color: #ff0000"
                     /translation="VVMEGSVNGHQFKCTGEGEGNPYMGTQTMRIKVIEGGPLPFAFDI
                     LATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGVVTVMQDTSLEDGCLV
                     YHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHMALKVDGGGHLSCSFV
                     TTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVAKFAGLGGGMDELYK"
     primer_bind     3736..3755
                     /label=oSEQ062 mRuby2_fwd
                     /note="color: black; sequence: acggccaccaattcaaatgc; added:
                     2025-02-27"
     primer_bind     complement(3736..3755)
                     /label=oSEQ093 mRuby2_rev
                     /note="color: black; sequence: gcatttgaattggtggccgt; added:
                     2025-02-27"
     primer_bind     complement(4268..4287)
                     /label=oSEQ061 mRuby2_rev
                     /note="color: black; sequence: cctctaacctttccaggcgg; added:
                     2025-02-27"
     primer_bind     4302..4321
                     /label=OSEQ091 mRuby2_fwd_SEQ
                     /note="color: black; sequence: atgttcgtagtacaacgcga; added:
                     2025-02-27"
     misc_feature    4394..4415
                     /label=miR-FF6 Target Site
                     /note="color: #cc99ff"
     polyA_signal    4433..4657
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3; direction: RIGHT"
     primer_bind     complement(4640..4656)
                     /label=oSEQ078 bGH_rev
                     /note="color: black; sequence: catagagcccaccgcat; added: 
                     2025-02-27"
ORIGIN
        1 cgctagtaga ctgcaggtgg gcccgagctt aagtaactaa ctaacaggaa gagtttgtag
       61 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
      121 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
      181 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
      241 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgctt
      301 agtagttaga cgtccccgag atccatgcta gcggtaatac ggttatccac agaatcaggg
      361 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
      421 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
      481 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      541 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
      601 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
      661 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
      721 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
      781 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
      841 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
      901 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
      961 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1021 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cggggcccaa
     1081 tctgaataat gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     1141 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     1201 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     1261 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     1321 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     1381 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     1441 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagctagacg aaatacgcga
     1501 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     1561 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     1621 tttccgggga tcgcagtggt gagtaaccat gcatcaccag gagtacggat aaaatgcttg
     1681 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     1741 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     1801 tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     1861 tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg ttgaatatgg
     1921 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     1981 atatttttat cttgtgcaat gtaacatcag agattttgag acacgggcac ctgcagtctt
     2041 cgtactgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     2101 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
     2161 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     2221 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
     2281 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     2341 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     2401 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     2461 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     2521 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     2581 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     2641 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     2701 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     2761 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     2821 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     2881 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     2941 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     3001 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt
     3061 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     3121 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     3181 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaga gctcgtttag
     3241 tgaaccgtca gatcgcctgg agacgccatc cacgctgtaa tggccaccat ggtgtctaag
     3301 ggcgaagagc tgatcaagga aaatatgcgt atgaaggtga gtatgtgctc gcttcggcag
     3361 cacatatact atgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt
     3421 ggaaacactt gctgggatta cttcttcagg ttaacccaac agaaggctcg agtgctgttg
     3481 acagtgagcg ccgcttgaag tctttaatta aatagtgaag ccacagatgt atttaattaa
     3541 agacttcaag cggtgcctac tgcctcggag aattcaaggg gctactttag gagcaattat
     3601 cttgtttact aaaactgaat accttgctat ctctttgata catttttaca aagctgaatt
     3661 aaaatggtat aaattaaatc acttttttca attgtttcct tttttttcct caggtggtca
     3721 tggaaggttc ggtcaacggc caccaattca aatgcacagg tgaaggagaa ggcaatccgt
     3781 acatgggaac tcaaaccatg aggatcaaag tcatcgaggg aggacccctg ccatttgcct
     3841 ttgacattct tgccacgtcg ttcatgtatg gcagccgtac ttttatcaag tacccgaaag
     3901 gcattcctga tttctttaaa cagtcctttc ctgagggttt tacttgggaa agagttacga
     3961 gatacgaaga tggtggagtc gtcaccgtca tgcaggacac cagccttgag gatggctgtc
     4021 tcgtttacca cgtccaagtc agaggggtaa actttccctc caatggtccc gtgatgcaga
     4081 agaagaccaa gggttgggag cctaatacag agatgatgta tccagcagat ggtggtctga
     4141 ggggatacac tcatatggca ctgaaagttg atggtggtgg ccatctgtct tgctctttcg
     4201 taacaactta caggtcaaaa aagaccgtcg ggaacatcaa gatgcccggt atccatgccg
     4261 ttgatcaccg cctggaaagg ttagaggaaa gtgacaatga aatgttcgta gtacaacgcg
     4321 aacacgcagt tgccaagttc gccgggcttg gtggtgggat ggacgagctg tacaagtaac
     4381 aacaagcttg tggaaccaaa gagattcctc ataaacctag gaggagttgc acctgtgcct
     4441 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     4501 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     4561 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     4621 aatagcaggc atgctgggga tgcggtgggc tctatgg
//
