LOCUS       sequence-406187-        5160 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 5160)
  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 5160)
  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 puro hPGK
FEATURES             Location/Qualifiers
     source          1..5160
                     /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"
     CDS             2074..2673
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2074..2093)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2570..2590
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2833..2954
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2870..2889)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2924..2943
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3003..3019)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3003..3019)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3016..3038)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3027..3043
                     /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(3051..3057,3058..3075,3076..3081))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3096..3117
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3234..3251)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3405..3993)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3485..3504)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4164..4955,4956..5024))
                     /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     4787..4806
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5025..5129)
                     /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 accatgaccg agtacaagcc cacggtgcgc
     2101 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     2161 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     2221 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     2281 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     2341 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     2401 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga
     2461 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     2521 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     2581 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2641 acctggtgca tgacccgcaa gcccggtgcc tgattcgaaa tgaccgacca agcgacgccc
     2701 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     2761 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     2821 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     2881 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     2941 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     3001 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3061 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3121 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3181 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3241 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3301 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3361 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3421 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3481 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3541 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3601 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3661 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3721 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3781 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3841 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3901 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3961 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4021 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4081 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4141 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4201 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4261 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4321 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4381 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4441 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4501 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4561 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4621 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4681 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4741 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4801 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4861 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4921 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4981 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5041 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5101 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
