LOCUS       sequence-406189-        5544 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 5544)
  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 5544)
  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 hPGK
FEATURES             Location/Qualifiers
     source          1..5544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        234..734
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     653..671
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     primer_bind     complement(1003..1020)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1009..1233
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1279..1707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1366..1385)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1576..1597
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1716..1736)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1721..2050
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1901..2036
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1963..1982
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    3217..3338
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3254..3273)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3308..3327
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3387..3403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3387..3403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3400..3422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3411..3427
                     /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(3435..3441,3442..3459,3460..3465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3480..3501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3618..3635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3789..4377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3869..3888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4548..5339,5340..5408))
                     /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     5171..5190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5409..5513)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgtttgcgcc
      241 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
      301 aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc
      361 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
      421 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctta
      481 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
      541 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
      601 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
      661 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
      721 acctctctcc ccaggctagc gtttaaactt aagcttggta ccgagctcgg atccactagt
      781 ccagtgtggt ggaattcaaa gtgctgagat tacaggcgtg agccaccacc cccggcccac
      841 tttttgtaaa ggtacgtact aatgactttt tttttatact tcagggagac ggttgtgtac
      901 gtctcggtaa gaagcaagga aaagaattag gctcggcacg gtagctcaca cctgtaatcc
      961 cagcactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     1021 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     1081 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     1141 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     1201 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     1261 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1321 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1381 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     1441 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1501 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1561 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1621 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1681 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     1741 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     1801 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     1861 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     1921 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     1981 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2041 cttttgcaaa aagctcccgg gaccggtgcc accatgaaaa agcctgaact caccgcgacg
     2101 tctgtcgaga agtttctgat cgaaaagttc gacagcgtgt ccgacctgat gcagctctcg
     2161 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     2221 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     2281 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     2341 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     2401 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     2461 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     2521 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     2581 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     2641 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     2701 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     2761 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     2821 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     2881 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     2941 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     3001 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtacttcg ccgatagttc
     3061 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     3121 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     3181 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     3241 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     3301 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     3361 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3421 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     3481 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3541 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3601 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3661 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3721 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3781 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3841 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3901 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3961 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4021 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4081 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4141 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4201 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     4261 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4321 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4381 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4441 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4501 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4561 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4621 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4681 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4741 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4801 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4861 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4921 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4981 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5041 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5101 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5161 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5221 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5281 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5341 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5401 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5461 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5521 ccccgaaaag tgccacctga cgtc
//
