LOCUS       sequence-406162-        5572 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 5572)
  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 5572)
  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
FEATURES             Location/Qualifiers
     source          1..5572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     304..323
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(444..773)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      458..593
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(512..531)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     758..778
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(787..1215)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(897..918)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1109..1128
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1261..1485
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1474..1491
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(1772..1791)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1773..1791)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1836..2039)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1865..1885)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2040..2419
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2591..2610
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2685..2789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2790..2858,2859..3650)
                     /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(3008..3027)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3821..4409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4310..4329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4563..4580
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4697..4718
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4733..4738,4739..4756,4757..4763)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4771..4787
                     /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     4776..4798
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4795..4811
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4795..4811
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4860..4981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4871..4890)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4925..4944
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5155..5572,1..377))
                     /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(5266..5285)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
       61 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
      121 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
      181 cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
      241 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
      301 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
      361 ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga tccgaaaatg
      421 gatatacaag ctcccgggag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac
      481 tacttctgga atagctcaga ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt
      541 agtcagccat ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga
      601 gttaggggcg ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
      661 gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc
      721 atacttctgc ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac
      781 agaattaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
      841 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
      901 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
      961 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     1021 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     1081 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     1141 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     1201 cgctacaggg cgcgtgggga taccccctag agccccagct ggttctttcc gcctcagaag
     1261 ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc
     1321 tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa
     1381 tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca
     1441 cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc
     1501 gggtttaaac gggccctcta gactcgagtg ctgggattac aggtgtgagc taccgtgccg
     1561 agcctaattc ttttccttgc ttcttaccga gacgtacaca accgtctccc tgaagtataa
     1621 aaaaaaagtc attagtacgt acctttacaa aaagtgggcc gggggtggtg gctcacgcct
     1681 gtaatctcag cactttgaat tccaccacac tggactagtg gatccgagct cggtaccaag
     1741 cttaagttta aacgctagcc agcttgggtc tccctatagt gagtcgtatt aatttcgata
     1801 agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata gacctcccac
     1861 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     1921 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     1981 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     2041 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     2101 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     2161 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     2221 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     2281 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     2341 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     2401 ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa
     2461 cgcctaaccc taagcagatt cttcatgcaa ttgtcggtca agccttgcct tgttgtagct
     2521 taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag cagatactgg
     2581 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg atcgggagat
     2641 ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     2701 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2761 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2821 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2881 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2941 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3001 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3061 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3121 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3181 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3241 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3301 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3361 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3421 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3481 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3541 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3601 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3661 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3721 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3781 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3841 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3901 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3961 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4021 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4081 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4141 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4201 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4261 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4321 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4381 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4441 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4501 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4561 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     4621 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4681 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     4741 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     4801 acagctatga ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat
     4861 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     4921 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     4981 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     5041 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     5101 gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa
     5161 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     5221 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     5281 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     5341 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     5401 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     5461 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     5521 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat ca
//
