LOCUS       sequence-406173-        4871 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 4871)
  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 4871)
  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 EF1a
FEATURES             Location/Qualifiers
     source          1..4871
                     /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..445
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     complement(714..731)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    720..944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      990..1418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1077..1096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1287..1308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1427..1447)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1432..1761
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1612..1747
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1674..1693
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1785..2384
                     /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(1785..1804)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2281..2301
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2544..2665
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2581..2600)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2635..2654
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2714..2730)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2714..2730)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2727..2749)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2738..2754
                     /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(2762..2768,2769..2786,2787..2792))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2807..2828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2945..2962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3116..3704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3196..3215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3875..4666,4667..4735))
                     /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     4498..4517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4736..4840)
                     /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 cgtgggcaga
      241 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc
      301 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      361 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
      421 caacgggttt gccgccagaa cacaggctag cgtttaaact taagcttggt accgagctcg
      481 gatccactag tccagtgtgg tggaattcaa agtgctgaga ttacaggcgt gagccaccac
      541 ccccggccca ctttttgtaa aggtacgtac taatgacttt ttttttatac ttcagggaga
      601 cggttgtgta cgtctcggta agaagcaagg aaaagaatta ggctcggcac ggtagctcac
      661 acctgtaatc ccagcactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
      721 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
      781 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
      841 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
      901 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
      961 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1021 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1081 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1141 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1201 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1261 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1321 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1381 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     1441 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     1501 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     1561 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     1621 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     1681 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     1741 ggaggcctag gcttttgcaa aaagctcccg ggaccggtgc caccatgacc gagtacaagc
     1801 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     1861 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     1921 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     1981 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     2041 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     2101 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     2161 ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     2221 ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc
     2281 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     2341 aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgattcgaa atgaccgacc
     2401 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     2461 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     2521 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     2581 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2641 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     2701 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2761 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2821 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2881 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2941 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3001 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3061 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3121 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3181 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3241 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3301 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3361 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3421 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3481 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3541 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3601 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3661 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3721 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3781 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3841 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3901 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3961 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4021 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4081 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4141 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4201 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4261 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4321 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4381 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4441 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4501 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4561 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4621 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4681 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4741 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4801 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4861 cacctgacgt c
//
