LOCUS       sequence-181300-        8719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Laccase2 circular RNA when the upstream SV40 poly(A) 
            signal fails to be used.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8719)
  AUTHORS   Liang D, Tatomer DC, Wilusz JE
  TITLE     Use of circular RNAs as markers of readthrough transcription to 
            identify factors regulating cleavage/polyadenylation events.
  JOURNAL   Methods. 2021 Apr 18. pii: S1046-2023(21)00104-3. doi: 
            10.1016/j.ymeth.2021.04.012.
  PUBMED    33882363
REFERENCE   2  (bases 1 to 8719)
  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: "Methods. 
            2021 Apr 18. pii: S1046-2023(21)00104-3. doi: 
            10.1016/j.ymeth.2021.04.012."
COMMENT     Sequence Label: pCRII-TOPO CMV-cGFP-SV40 pA + Laccase2 Exon 2
FEATURES             Location/Qualifiers
     source          1..8719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(943..1734,1735..1803))
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(1821..2615)
                     /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(1932..1951)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2542..2561
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(2959..3387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3069..3090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3281..3300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3513..3535
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3527..3544
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3528..3544
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3551..3570
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3551..3569
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6172..6293
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6183..6202)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6237..6256
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6302..7021)
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MASKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     promoter        complement(7065..7268)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7094..7114)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7269..7647
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        complement(8465..8483)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(8466..8483)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(8501..8517)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8501..8517)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8514..8536)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8525..8541
                     /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(8549..8555,8556..8573,8574..8579))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8594..8615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     1861 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     1921 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     1981 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     2041 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     2101 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     2161 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     2221 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     2281 ttctcggcag gagcaaggtg ggatgacagg agatcctgcc ccggcacttc gcccaatagc
     2341 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     2401 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     2461 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     2521 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     2581 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     2641 tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
     2701 actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt
     2761 gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc
     2821 tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg
     2881 ggtcagcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg
     2941 cgccctgaat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3001 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3061 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3121 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3181 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3241 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3301 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3361 cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga
     3421 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     3481 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     3541 cagtgaattg taatacgact cactataggg cgaattgggc cctctagatg catgctcgag
     3601 agtttgtaca ttttgccctt agctttttgt ttcctagctt gtcttttttc ttctgcttcc
     3661 tacttttcag gtttaaattt atcttttttc ttctaaaagt atgtttttat cttctaattt
     3721 ccctatcttc tctattcttt tcttcgcctt cccgtacttc tgtcttccag ttttccactt
     3781 caaacttcta tcttctccaa attgtttcat cctaccactc ccaattaatc tttccatttt
     3841 cgtctgcgtt tagtaaatgc gttaactagg ctttaaatga cgcaattctc cctgcgtcat
     3901 ggatttcaag gtcttttaat caccttcggt ttaatctctt tttaaaagat cgccttcaaa
     3961 ttattttaat cacctacaac ttttaaacta aactttaagc tgtttaagtc accttcattt
     4021 taatctaaaa gcattgccct tctattggta ttaattcggg gctctgtagt cctttctctc
     4081 aattttcttt taaatacatt ttttactcca tgaagaagct tcatctcaac ctccgtcatg
     4141 ttttagaaac cttttatctt ttccttcctc atgctactct tctaagtctt catattttct
     4201 cttaaaatct taagcccaaa taaatattca tattaaataa aaagctataa tcgatattca
     4261 tatgattaaa ttcattttca ttgtatataa aaaataagtc gatttcccca actatcagat
     4321 actcgttact cagctagtgt gaatgcgaac gcgaaattta ataatttcat aatcggggat
     4381 atcgatagat attggggaat aaaatgagaa aaaatttgat aattgctcaa tagtgcgggc
     4441 gtgaccaaat tggcggcttt agggcgttaa cgcgtgacaa aaagtttttt ggcaaattga
     4501 tggaaattta cacgagtaat acaattatga aaaaatttcg aaaatagttc aaaaatgtgg
     4561 gcatggcagt tttgggcaat ttgagaaagt taaagcgtgg cagcatggat caacaaactt
     4621 gctctgcgtc cttgtctcta gaatctgtat gctaaaactc atccttctag cttttatagt
     4681 tcctgatgtc tcggcggtaa taaaacaata agacagacgg gcatggcgag atcgactcgg
     4741 ctattgatcc ttattaagaa tatatatact ttacatggtc ggaaatgctt tattcttcct
     4801 gttacatact tttcaacgaa tctgcaatag tatacaatag tatagaaact gcctattaaa
     4861 aaatcgaata tttctagtaa aaaattttgg aattttgaat acttacgctc ccaatactgt
     4921 gtagtattcc aacgtaaaat gatagtagca aattctcgga ggctcgcctt cgcgacaagc
     4981 gcgtgcacat tcatcaggag cagatagcgc aggattgcgc cgcagctcgg cagttgcact
     5041 agtagagaaa tctaagtggc ggaatggaga ttttgggttg ggttccacga cggttcctgt
     5101 gatcttatta gctggataac gtgcagaggt cggtagactc ccgcgtgagt ttaggtaacc
     5161 gcttccagcg gttagggctg gcttagtacc agctaggatt gaggatggag ctccagaccc
     5221 aaggctattg ccatttaatg gagcgggccc agagtggctt ccacctatag acgggtgagt
     5281 ctgcacaagg ccgtgtgttg ccgaaaaaaa tggtgaatcg aatggagttg aactactagg
     5341 agtggccaca tgttgctgcc agaaggaccc cattggtgta agatctggtt gattttctgc
     5401 ataaaataaa aaaaatatat ataacaataa aacttagtct cgatttagct tattattata
     5461 cccataaatc gtacagtaaa aaggtatact agattcgttg aaaaataggt aacatgtaga
     5521 aggaagcggt ataaagtata tatattcttg atcaggatca aagcctagtc gatgtaaccc
     5581 tgtcctcgcc tactctaacg ccctaaaacc gcccaaaact gtcacgccca cgtgtttaaa
     5641 attttgttgt attttttcat aattttataa gtcttgtaaa tttctagcga tttgccaaaa
     5701 aactttttgc cacgccctaa acccgcccaa aatttattta tttaaattta ttccccaata
     5761 tctatcgata tccgagaaaa ctgataaaat tttgggttcg cattccccct agataagtaa
     5821 tgggtatcag atagtcggga aactatacta tacgactata gcattctctc ttgttttttc
     5881 tttttgtagc ttactggctt cgcaacagaa tttaatgttt ttgaagtcgt tattacagag
     5941 accaaataaa agtcgacaaa gatcaatgac ttgagagcac cggaactcag tcatttctca
     6001 atggctaagc aatatcttag taacctattg actatatttt ttaagtagtt gtattaatct
     6061 ctatctttca aagagaaaaa aacttatctg cggtttcctc aagctccgcc tgccccctca
     6121 gcagaagccc ctgcgctggc tgcctcaatg cctaccgcac agcgcggccg ctaagataca
     6181 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     6241 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttgcggccg
     6301 ctcacttgta cagctcatcc atgccgtggg tgatggcggc ggcggtcacg aagccgaagt
     6361 agatcatgtg atcgcgcttc tcgttggggt ccttggacag ggcgctctgg gtggacaggt
     6421 agtggttatc gggcagcagc acagggccat cgccgatggg ggtattctgc tggtagtggt
     6481 cggccagctg cacgctgcca tcctcgatgt tgtggcggat cttgaagttc accttgatgc
     6541 cattcttggc cttgtcggtc atgatgtaca cattgtgggc gttgtagttg tactccatct
     6601 tattgcccag gatgttgcca tcctccttga aatcggtgcc ggtcagctcg atgcgattca
     6661 ccagggtatc gccctcgaac ttcacctcgg cgcgcgactt gtagttgccg tcatcctcga
     6721 agaagatggt gcgctcctgg atgtagccct caggcatggc gctcttgaag aagtcgtgct
     6781 gcttcatgtg atcggggtag cgtgagaagc actgcacgcc gtagctcagg gtggtcacca
     6841 gggtgggcca gggcacaggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc
     6901 cgtaggtggc atcgccctcg ccctcgccgc tcacgctgaa cttgtggcca ttcacatcgc
     6961 cattcagctc gatcaggatg ggcacgatgc cggtgaacag ctcggcgccc ttgctggcca
     7021 tggtgatatc gccagtaagc agtgggttct ctagttagcc agagagctct gcttatatag
     7081 acctcccacc gtacacgcct accgcccatt tgcgtcaatg gggcggagtt gttacgacat
     7141 tttggaaagt cccgttgatt ttggtgccaa aacaaactcc cattgacgtc aatggggtgg
     7201 agacttggaa atccccgtga gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac
     7261 cgcatcacca tggtaatagc gatgactaat acgtagatgt actgccaagt aggaaagtcc
     7321 cataaggtca tgtactgggc ataatgccag gcgggccatt taccgtcatt gacgtcaata
     7381 gggggcgtac ttggcatatg atacacttga tgtactgcca agtgggcagt ttaccgtaaa
     7441 tactccaccc attgacgtca atggaaagtc cctattggcg ttactatggg aacatacgtc
     7501 attattgacg tcaatgggcg ggggtcgttg ggcggtcagc caggcgggcc atttaccgta
     7561 agttatgtaa cgcggaactc catatatggg ctatgaacta atgaccccgt aattgattac
     7621 tattaataac tagtcaataa tcaatgtgga tccgacggag aacaactcgc atcaccggaa
     7681 ttcgatcacc ttccgccgcc tttgtgaggg aggcaaaaag tagttcttcg gttaaaaata
     7741 ggttctagtt ttacttttct agatttttct taacagctta tggaacttga atgcaaacta
     7801 cacatgcaga aatacttaaa ttactagcgt gtggaaagat ttgagctgca aactttagac
     7861 aaaagaaaca tggaaatcat attttaaacg ggtcatcaaa cacctcacaa aacccccgga
     7921 acttttaaaa tacctcttaa agcacttctt gtgttctctt gagggacagt aggtatagtt
     7981 taccaccttt tgaaggaaga ttaaagtgtg atagttcagg gctttacttt ccattacgca
     8041 actgagcccc agccttgcag ggacggttga gaagtggcaa aatggcggac tttctccccc
     8101 aactgcttgc agtcctgcga cttgctccca agacagccac acgttttgct tttttgttcg
     8161 agaaatcgga gcagcacggg ctgtctgctt gggaaatctt agaaacgtga aaacccactc
     8221 ttggaaaacg cctcaatccc acaccacaga gctgctcccc gcctgagccc cgggggccgt
     8281 tggctgccag tggcccactc tgatctgcgg ccccccagtt gccgccagcc ccccacggcc
     8341 cgcacggaaa tttttctacc gtttttcagc ttccaggctc tcctgaaagt gctcacaagg
     8401 caaatcgcca tggaaagcga gttcaagtgg ccttttaaag aagcttgatg catagcttga
     8461 gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     8521 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     8581 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     8641 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     8701 gcggtttgcg tattgggcg
//
