LOCUS       sequence-406166-        5119 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 5119)
  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 5119)
  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
FEATURES             Location/Qualifiers
     source          1..5119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      191..200
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(218..547)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      232..367
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(286..305)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     532..552
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(561..989)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(671..692)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     883..902
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1035..1259
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1248..1265
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(1546..1565)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1547..1565)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1610..1813)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1639..1659)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1814..2193
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2365..2384
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2459..2563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2564..2632,2633..3424)
                     /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     complement(2782..2801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3595..4183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4084..4103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4337..4354
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4471..4492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4507..4512,4513..4530,4531..4537)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4545..4561
                     /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)."
     primer_bind     4550..4572
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4569..4585
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4569..4585
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4634..4755
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4645..4664)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4699..4718
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(4915..5119,1..194))
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
ORIGIN
        1 gcagtgccca gcaccacgag ttctgcacaa ggtcccccag taaaatgata tacattgaca
       61 ccagtgaaga tgcggccgtc gctagagaga gctgcgctgg cgacgctgta gtcttcagag
      121 atggggatgc tgttgattgt agccgttgct ctttcaatga gggtggattc ttcttgagac
      181 aaaggcttgg ccatggtggc accggtcccg ggagcttttt gcaaaagcct aggcctccaa
      241 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat
      301 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
      361 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
      421 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
      481 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
      541 gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa
      601 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
      661 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
      721 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
      781 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
      841 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
      901 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
      961 cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct
     1021 ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca
     1081 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     1141 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     1201 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     1261 cgaggctgat cagcgggttt aaacgggccc tctagactcg agtgctggga ttacaggtgt
     1321 gagctaccgt gccgagccta attcttttcc ttgcttctta ccgagacgta cacaaccgtc
     1381 tccctgaagt ataaaaaaaa agtcattagt acgtaccttt acaaaaagtg ggccgggggt
     1441 ggtggctcac gcctgtaatc tcagcacttt gaattccacc acactggact agtggatccg
     1501 agctcggtac caagcttaag tttaaacgct agccagcttg ggtctcccta tagtgagtcg
     1561 tattaatttc gataagccag taagcagtgg gttctctagt tagccagaga gctctgctta
     1621 tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac
     1681 gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg
     1741 ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc
     1801 aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
     1861 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
     1921 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
     1981 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
     2041 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     2101 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
     2161 attactatta ataactagtc aataatcaat gtcaacgcgt atatctggcc cgtacatcgc
     2221 gaagcagcgc aaaacgccta accctaagca gattcttcat gcaattgtcg gtcaagcctt
     2281 gccttgttgt agcttaaatt ttgctcgcgc actactcagc gacctccaac acacaagcag
     2341 ggagcagata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     2401 ggggatcggg agatctcccg atccgtcgac gtcaggtggc acttttcggg gaaatgtgcg
     2461 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2521 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     3001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     3061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     3121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     3181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     3241 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     3301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3541 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3601 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3661 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3721 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     3781 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3841 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3901 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3961 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     4021 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     4081 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     4141 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     4201 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     4261 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     4321 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     4381 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     4441 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     4501 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     4561 atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag aggtcgacgg
     4621 tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     4681 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     4741 tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
     4801 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
     4861 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaattagcc
     4921 ctcccacaca taaccagagg gcagcaattc acgaatccca actgccgtcg gctgtccatc
     4981 actgtccttc actatggctt tgatcccagg atgcagatcg agaagcacct gtcggcaccg
     5041 tccgcagggg ctcaagatgc ccctgttctc atttccgatc gcgacgatac aagtcaggtt
     5101 gccagctgcc gcagcagca
//
