LOCUS       sequence-406165-        5704 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 5704)
  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 5704)
  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 hygro
FEATURES             Location/Qualifiers
     source          1..5704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     363..382
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     616..633
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    750..771
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(786..791,792..809,810..816)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    824..840
                     /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     829..851
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     848..864
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     848..864
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    913..1034
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(924..943)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     978..997
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2201..2530)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2215..2350
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2269..2288)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2515..2535
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2544..2972)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2654..2675)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2866..2885
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3018..3242
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3231..3248
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3529..3548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3530..3548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3593..3796)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3622..3642)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3797..4176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4348..4367
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4442..4546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4547..4615,4616..5407)
                     /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(4765..4784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5578..5704,1..462)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
       61 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
      121 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
      181 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
      241 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
      301 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
      361 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
      421 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
      481 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
      541 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
      601 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      661 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      721 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      781 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      841 tttcacacag gaaacagcta tgaccatgat tacgccaagc tctagctaga ggtcgacggt
      901 atacagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
      961 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     1021 gcaataaaca agttggggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc
     1081 atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg
     1141 gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaactatcgg
     1201 cgaagtactt ctacacagcc atcggtccag acggccgcgc ttctgcgggc gatttgtgta
     1261 cgcccgacag tcccggctcc ggatcggacg attgcgtcgc atcgaccctg cgcccaagct
     1321 gcatcatcga aattgccgtc aaccaagctc tgatagagtt ggtcaagacc aatgcggagc
     1381 atatacgccc ggagccgcgg cgatcctgca agctccggat gcctccgctc gaagtagcgc
     1441 gtctgctgct ccatacaagc caaccacggc ctccagaaga agatgttggc gacctcgtat
     1501 tgggaatccc cgaacatcgc ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc
     1561 gtcaggacat tgttggagcc gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc
     1621 tcggcccaaa gcatcagctc atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc
     1681 atcacagttt gccagtgata cacatgggga tcagcaatcg cgcatatgaa atcacgccat
     1741 gtagtgtatt gaccgattcc ttgcggtccg aatgggccga acccgctcgt ctggctaaga
     1801 tcggccgcag cgatcgcatc catggcctcc gcgaccggct gcagaacagc gggcagttcg
     1861 gtttcaggca ggtcttgcaa cgtgacaccc tgtgcacggc gggagatgca ataggtcagg
     1921 ctctcgctga attccccaat gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag
     1981 tgccgataaa cataacgatc tttgtagaaa ccatcggcgc agctatttac ccgcaggaca
     2041 tatccacgcc ctcctacatc gaagctgaaa gcacgagatt cttcgccctc cgagagctgc
     2101 atcaggtcgg acacgctgtc gaacttttcg atcagaaact tctcgacaga cgtcgcggtg
     2161 agttcaggct ttttcatggt ggcaccggtc ccgggagctt tttgcaaaag cctaggcctc
     2221 caaaaaagcc tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg
     2281 cataaataaa aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta
     2341 ggggcgggat gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc
     2401 tttgcatact tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat
     2461 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     2521 actgacacac attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt
     2581 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2641 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2701 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2761 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2821 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2881 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2941 cgccgcgctt aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt
     3001 tctttccgcc tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc
     3061 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
     3121 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
     3181 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     3241 agtcgaggct gatcagcggg tttaaacggg ccctctagac tcgagtgctg ggattacagg
     3301 tgtgagctac cgtgccgagc ctaattcttt tccttgcttc ttaccgagac gtacacaacc
     3361 gtctccctga agtataaaaa aaaagtcatt agtacgtacc tttacaaaaa gtgggccggg
     3421 ggtggtggct cacgcctgta atctcagcac tttgaattcc accacactgg actagtggat
     3481 ccgagctcgg taccaagctt aagtttaaac gctagccagc ttgggtctcc ctatagtgag
     3541 tcgtattaat ttcgataagc cagtaagcag tgggttctct agttagccag agagctctgc
     3601 ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt
     3661 tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa
     3721 tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact
     3781 gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag
     3841 gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga
     3901 cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt
     3961 accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa
     4021 catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat
     4081 ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa
     4141 ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat
     4201 cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc
     4261 cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag
     4321 cagggagcag atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4381 ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt
     4441 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4501 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4561 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4621 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4681 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4741 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4801 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4861 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4921 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4981 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5041 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5101 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5161 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5221 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5281 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5341 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5401 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5461 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5521 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5581 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5641 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5701 caga
//
