LOCUS       sequence-374522-        6097 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for generating template plasmids to transcribe 
            circRNA, with mNeonGreen dropout following BsaI digestion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1815 to 1849)
  AUTHORS   Chen R, Wang SK, Belk JA, Amaya L, Li Z, Cardenas A, Abe BT, Chen 
            CK, Wender PA, Chang HY
  TITLE     Engineering circular RNA for enhanced protein production.
  JOURNAL   Nat Biotechnol. 2022 Jul 18. pii: 10.1038/s41587-022-01393-0. doi: 
            10.1038/s41587-022-01393-0.
  PUBMED    35851375
REFERENCE   2  (bases 1 to 6097)
  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: "Nat 
            Biotechnol. 2022 Jul 18. pii: 10.1038/s41587-022-01393-0. doi: 
            10.1038/s41587-022-01393-0."
COMMENT     Sequence Label: pRC0940 (circRNA entry vector)
FEATURES             Location/Qualifiers
     source          1..6097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          646..1662
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1586..1608
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1670..1689
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1732..1751
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1732..1750
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1815..1849
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             1858..1869
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2612..2683
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2699..2726
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2799..2846
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2860..3084
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3127..3143
                     /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     3132..3154
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3151..3167
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3151..3167
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3216..3337
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3227..3246)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3281..3300
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3497..4096)
                     /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(3580..3600)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4077..4096
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4107..4435)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4121..4256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4175..4194)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             complement(4469..5284)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4469..5284)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5192..5211
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5276..5295
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5509..6097
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5998..6017
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
       61 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      121 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      181 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      241 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      301 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      361 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
      421 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
      481 gggggggggg ggcgcscrgg gggggggggg gggggggggg gggggggggg ggsgaggcgg
      541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      661 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      721 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      781 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
      841 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
      901 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
      961 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     1021 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     1081 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     1141 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     1201 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     1261 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     1321 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     1381 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     1441 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     1501 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     1561 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     1621 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     1681 gttattgtgc tgtctcatca ttttggcaaa gcggccgcgg ccagtgaatt gtaatacgac
     1741 tcactatagg ggttaacaaa aaaaaaaaaa aaaaaaaaaa atcaccgacc gatctatctg
     1801 aaccggttga gaccttgacg gctagctcag tcctaggtac agtgctagct actagagaaa
     1861 gaggagaaat actagatggt cagcaaaggc gaagaagaca acatggcatc actgccagcg
     1921 acacacgaac ttcatatttt cggctccatc aatggtgtgg acttcgatat ggttggacag
     1981 ggtactggga accctaacga tggatatgag gagttgaact tgaagagtac caagggcgat
     2041 cttcagtttt caccttggat tctcgtacct catataggtt atggctttca tcaatatctg
     2101 ccctatcctg acggtatgtc acctttccaa gcagctatgg ttgacgggag cggttatcag
     2161 gttcaccgga caatgcagtt tgaagatgga gcctctttga ccgtaaatta tcggtatact
     2221 tatgagggca gccatattaa aggcgaggct caagttaagg ggactgggtt ccccgcggat
     2281 ggtcccgtaa tgacaaacag tctgacggct gcagactggt gccgctctaa gaagacatat
     2341 ccaaatgaca agacgattat tagcactttc aagtggtcat acactacagg aaatggtaaa
     2401 cgctatagga gcactgcccg gaccacgtat actttcgcga agccgatggc ggcaaattat
     2461 ttgaaaaacc aaccgatgta tgtgttcaga aagacagaac tcaagcattc taaaactgaa
     2521 ctgaacttca aagagtggca gaaagccttt actgatgtaa tgggcatgga cgaactctat
     2581 aagtaataac caggcatact agtccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     2641 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg
     2701 ctcaccttcg ggtgggcctt tctgcgttta taggtctcaa cgcgttcaga tagatcggtc
     2761 ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa gcggccgcct agcataaccc cttggggcct
     2821 ctaaacgggt cttgaggggt tttttggcta gcggccggcc tgtgccttct agttgccagc
     2881 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2941 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3001 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3061 ctggggatgc ggtgggctct atggcggcgc gccactttat gcttccggct cgtatgttgt
     3121 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     3181 agctctagct agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca
     3241 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
     3301 tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca
     3361 tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca
     3421 acctttcata gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt
     3481 ggtcggtcat ttcgaatcag gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt
     3541 cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag gggaggttgc
     3601 ggggcgcgga ggtttccagg aaggcgggca ccccggcgcg ctcggccgcc tccactccgg
     3661 ggagcacgac ggcgctgccc agacccttgc cctggtggtc gggcgacacg ccgacggtgg
     3721 ccaggaacca cgcgggctcc ttgggccggt gcggcgccag gaggccttcc atctgttgct
     3781 gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg
     3841 cccccgcttc gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga
     3901 cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc agctcggtga
     3961 cccgctcgat gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg
     4021 cggcggcgag ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg
     4081 gcttgtactc ggtcatgcgg gagctttttg caaaagccta ggcctccaaa aaagcctcct
     4141 cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa
     4201 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
     4261 ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg
     4321 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt
     4381 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacagagct
     4441 cgtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact
     4501 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     4561 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     4621 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     4681 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca
     4741 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     4801 caaccaaacc gttattcatt cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt
     4861 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     4921 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     4981 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     5041 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     5101 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     5161 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     5221 cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc
     5281 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata
     5341 tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa
     5401 taaatcgaac ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt
     5461 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5521 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5581 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5641 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5701 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5761 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5821 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5881 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5941 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     6001 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6061 tttgtgatgc tcgtcagggg ggcggagcct atggaaa
//
