LOCUS       sequence-406185-        5412 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 5412)
  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 5412)
  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 hPGK
FEATURES             Location/Qualifiers
     source          1..5412
                     /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             2117..2911
                     /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(2171..2190)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2781..2800
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3085..3206
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3122..3141)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3176..3195
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3255..3271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3255..3271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3268..3290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3279..3295
                     /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(3303..3309,3310..3327,3328..3333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3348..3369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3486..3503)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3657..4245)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3737..3756)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4416..5207,5208..5276))
                     /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     5039..5058
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5277..5381)
                     /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 gagcttgtat atccattttc ggatctgatc aagagacagg
     2101 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2161 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2221 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2281 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2341 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2401 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2461 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2521 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2581 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     2641 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2701 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2761 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     2821 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     2881 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     2941 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3001 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3061 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3121 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3181 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3241 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3301 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3361 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3421 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3481 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3541 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3601 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3661 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3721 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3781 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3841 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3901 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3961 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4021 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4081 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4141 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4201 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4261 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4321 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4381 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4441 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4501 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4561 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4621 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4681 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4741 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4801 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4861 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4921 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4981 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5041 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5101 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5161 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5221 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5281 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5341 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5401 ccacctgacg tc
//
