LOCUS       sequence-406164-        5320 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 5320)
  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 5320)
  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
FEATURES             Location/Qualifiers
     source          1..5320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     275..294
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(318..647)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      332..467
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(386..405)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     632..652
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(661..1089)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(771..792)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     983..1002
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1135..1359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1348..1365
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(1646..1665)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1647..1665)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1710..1913)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1739..1759)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1914..2293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2465..2484
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2559..2663
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2664..2732,2733..3524)
                     /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(2882..2901)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3695..4283
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4184..4203
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4437..4454
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4571..4592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4607..4612,4613..4630,4631..4637)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4645..4661
                     /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     4650..4672
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4669..4685
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4669..4685
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4734..4855
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4745..4764)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4799..4818
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(5015..5320,1..294))
                     /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(5098..5118)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg
       61 cgtggtccag accgccaccg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag
      121 cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg
      181 atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc
      241 cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatggtggc
      301 accggtcccg ggagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
      361 tggaatagct cagaggcaga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
      421 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
      481 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
      541 gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt
      601 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccacagaatt
      661 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
      721 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
      781 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
      841 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
      901 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
      961 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     1021 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     1081 agggcgcgtg gggatacccc ctagagcccc agctggttct ttccgcctca gaagccatag
     1141 agcccaccgc atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct
     1201 gccccacccc accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct
     1261 cattttatta ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg
     1321 aggggcaaac aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgggttt
     1381 aaacgggccc tctagactcg agtgctggga ttacaggtgt gagctaccgt gccgagccta
     1441 attcttttcc ttgcttctta ccgagacgta cacaaccgtc tccctgaagt ataaaaaaaa
     1501 agtcattagt acgtaccttt acaaaaagtg ggccgggggt ggtggctcac gcctgtaatc
     1561 tcagcacttt gaattccacc acactggact agtggatccg agctcggtac caagcttaag
     1621 tttaaacgct agccagcttg ggtctcccta tagtgagtcg tattaatttc gataagccag
     1681 taagcagtgg gttctctagt tagccagaga gctctgctta tatagacctc ccaccgtaca
     1741 cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt
     1801 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
     1861 cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta
     1921 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac
     1981 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
     2041 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     2101 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     2161 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     2221 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtc
     2281 aataatcaat gtcaacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta
     2341 accctaagca gattcttcat gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt
     2401 ttgctcgcgc actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac
     2461 tatgcggcat cagagcagat tgtactgaga gtgcaccata ggggatcggg agatctcccg
     2521 atccgtcgac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2581 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2641 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2701 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2761 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2821 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2881 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2941 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3001 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3061 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3121 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3181 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3241 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3301 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3361 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3421 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3481 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3541 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3601 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3661 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3721 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3781 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3841 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3901 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3961 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4021 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4081 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4141 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4201 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4261 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4321 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4381 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4441 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     4501 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4561 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     4621 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     4681 atgaccatga ttacgccaag ctctagctag aggtcgacgg tatacagaca tgataagata
     4741 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     4801 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttggggt
     4861 gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga
     4921 aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg
     4981 gcaggttggg cgtcgcttgg tcggtcattt cgaatcaggc accgggcttg cgggtcatgc
     5041 accaggtgcg cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct
     5101 cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct
     5161 cggccgcctc cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg
     5221 gcgagactcc gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga
     5281 ggccttccat ctgttgctgc gcggccagcc gggaaccgct
//
