LOCUS       sequence-406172-        5123 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 5123)
  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 5123)
  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 neo EF1a
FEATURES             Location/Qualifiers
     source          1..5123
                     /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             1828..2622
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1882..1901)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2492..2511
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2796..2917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2833..2852)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2887..2906
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2966..2982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2966..2982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2979..3001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2990..3006
                     /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(3014..3020,3021..3038,3039..3044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3059..3080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3197..3214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3368..3956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3448..3467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4127..4918,4919..4987))
                     /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     4750..4769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4988..5092)
                     /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 ggagcttgta tatccatttt cggatctgat
     1801 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     1861 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     1921 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     1981 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     2041 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     2101 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     2161 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     2221 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     2281 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     2341 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     2401 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     2461 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     2521 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     2581 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     2641 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     2701 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     2761 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     2821 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2881 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     2941 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3001 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3061 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3121 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3181 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3241 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3301 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3361 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3421 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3481 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3541 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3601 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3661 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3721 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3781 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3841 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3901 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3961 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4021 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4081 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4141 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4201 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4261 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4321 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4381 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4441 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4501 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4561 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4621 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4681 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4741 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4801 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4861 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4921 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4981 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5041 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5101 cccgaaaagt gccacctgac gtc
//
