LOCUS       sequence-406180-        4670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expressing cloned exons as circular RNA. Alternatively, for 
            expressing cDNA (if the EcoRI-XhoI restriction fragment is also 
            removed)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4670)
  AUTHORS   Stringer BW, Gabryelska M, Marri S, Clark L, Lin H, Gantley L, Liu 
            R, Wilusz JE, Conn VM, Conn SJ
  TITLE     Versatile toolkit for highly-efficient and scarless overexpression 
            of circular RNAs.
  JOURNAL   bioRxiv. 2023 Nov 22:2023.11.21.568171. doi: 
            10.1101/2023.11.21.568171. Preprint.
  PUBMED    38045421
REFERENCE   2  (bases 1 to 4670)
  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"; journalName: "bioRxiv. 
            2023 Nov 22:2023.11.21.568171. doi: 10.1101/2023.11.21.568171. 
            Preprint."
COMMENT     Sequence Label: mc2 blast EF1a
FEATURES             Location/Qualifiers
     source          1..4670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(69..88)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        259..470
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(739..756)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    745..969
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1015..1443
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1102..1121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1312..1333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1452..1472)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1457..1786
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1637..1772
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1699..1718
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     regulatory      1804..1813
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1810..2208
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    2368..2489
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2405..2424)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2459..2478
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2538..2554)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2538..2554)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2551..2573)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2562..2578
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2586..2592,2593..2610,2611..2616))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2631..2652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2769..2786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2940..3528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3020..3039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3699..4490,4491..4559))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4322..4341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4560..4664)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
       61 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
      121 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
      181 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
      241 cgggccagat atacgcgtgg gcagagcgca catcgcccac agtccccgag aagttggggg
      301 gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      361 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      421 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gctagcgttt
      481 aaacttaagc ttggtaccga gctcggatcc actagtccag tgtggtggaa ttcaaagtgc
      541 tgagattaca ggcgtgagcc accacccccg gcccactttt tgtaaaggta cgtactaatg
      601 actttttttt tatacttcag ggagacggtt gtgtacgtct cggtaagaag caaggaaaag
      661 aattaggctc ggcacggtag ctcacacctg taatcccagc actcgagtct agagggcccg
      721 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
      781 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
      841 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
      901 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
      961 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     1021 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     1081 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     1141 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     1201 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1261 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1321 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1381 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1441 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     1501 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     1561 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     1621 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     1681 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     1741 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggacc
     1801 ggtgccacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     1861 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     1921 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     1981 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     2041 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     2101 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     2161 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaatt cgaaatgacc
     2221 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     2281 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     2341 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     2401 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2461 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     2521 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2581 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2641 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     2701 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     2761 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2821 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2881 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2941 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     3001 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     3061 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     3121 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     3181 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     3241 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     3301 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     3361 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     3421 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     3481 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3541 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3601 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3661 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3721 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3781 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3841 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3901 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3961 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     4021 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4081 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4141 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4201 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4261 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4321 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4381 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4441 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4501 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4561 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4621 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
//
