LOCUS       sequence-371630-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for generating template plasmids to transcribe mRNA, 
            with mNeonGreen dropout following BsaI digestion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 2395 to 2429)
  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 6172)
  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: mRNA-CleanCap entry vector
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        655..958
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1234..2250
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2174..2196
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2258..2277
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      2378..2387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        2395..2429
                     /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             2438..2449
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      3192..3263
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3279..3306
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      3463..3510
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    3524..3748
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3791..3807
                     /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     3796..3818
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3815..3831
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3815..3831
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3880..4001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3891..3910)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3945..3964
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4161..4760)
                     /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(4244..4264)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4741..4760
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4771..5099)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4785..4920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4839..4858)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             complement(5133..5948)
                     /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(5133..5948)
                     /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     5856..5875
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5940..5959
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaac ctgcaggtta
      601 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      661 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      721 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      781 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      841 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      901 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      961 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1021 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggs
     1081 gcgccrggsg gggggggggg gggsgggggg sggggsgggg sgrggcggrg aggtgcggcg
     1141 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     1201 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     1261 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     1321 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa
     1381 tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct
     1441 ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg
     1501 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg
     1561 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc
     1621 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg
     1681 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc
     1741 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg
     1801 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggrgggctcg
     1861 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc
     1921 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt
     1981 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg
     2041 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt
     2101 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     2161 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     2221 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     2281 tctcatcatt ttggcaaagc ggccgcggcc agtgaattgt aatacgactc actataagga
     2341 aataagagag aaaagaagag taagaagaaa tataagagcc accatggtga gaccttgacg
     2401 gctagctcag tcctaggtac agtgctagct actagagaaa gaggagaaat actagatggt
     2461 cagcaaaggc gaagaagaca acatggcatc actgccagcg acacacgaac ttcatatttt
     2521 cggctccatc aatggtgtgg acttcgatat ggttggacag ggtactggga accctaacga
     2581 tggatatgag gagttgaact tgaagagtac caagggcgat cttcagtttt caccttggat
     2641 tctcgtacct catataggtt atggctttca tcaatatctg ccctatcctg acggtatgtc
     2701 acctttccaa gcagctatgg ttgacgggag cggttatcag gttcaccgga caatgcagtt
     2761 tgaagatgga gcctctttga ccgtaaatta tcggtatact tatgagggca gccatattaa
     2821 aggcgaggct caagttaagg ggactgggtt ccccgcggat ggtcccgtaa tgacaaacag
     2881 tctgacggct gcagactggt gccgctctaa gaagacatat ccaaatgaca agacgattat
     2941 tagcactttc aagtggtcat acactacagg aaatggtaaa cgctatagga gcactgcccg
     3001 gaccacgtat actttcgcga agccgatggc ggcaaattat ttgaaaaacc aaccgatgta
     3061 tgtgttcaga aagacagaac tcaagcattc taaaactgaa ctgaacttca aagagtggca
     3121 gaaagccttt actgatgtaa tgggcatgga cgaactctat aagtaataac caggcatact
     3181 agtccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     3241 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     3301 tctgcgttta taggtctcat agtaatgagc tggagcctcg gtggccatgc ttcttgcccc
     3361 ttgggcctcc ccccagcccc tcctcccctt cctgcacccg tacccccgtg gtctttgaat
     3421 aaagtctgaa aaaaaaaaaa aaaaaaaaaa aaaagcggcc gcctagcata accccttggg
     3481 gcctctaaac gggtcttgag gggttttttg gctagcggcc ggcctgtgcc ttctagttgc
     3541 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3601 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3661 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3721 catgctgggg atgcggtggg ctctatggcg gcgcgccact ttatgcttcc ggctcgtatg
     3781 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     3841 gccaagctct agctagaggt cgacggtata cagacatgat aagatacatt gatgagtttg
     3901 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     3961 ttgctttatt tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc
     4021 agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag
     4081 cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc
     4141 gcttggtcgg tcatttcgaa tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt
     4201 ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg
     4261 ttgcggggcg cggaggtttc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact
     4321 ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga cacgccgacg
     4381 gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt
     4441 tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac
     4501 accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc
     4561 gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg
     4621 gtgacccgct cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg
     4681 aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc
     4741 gtgggcttgt actcggtcat gcgggagctt tttgcaaaag cctaggcctc caaaaaagcc
     4801 tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa
     4861 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     4921 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     4981 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     5041 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacag
     5101 agctcgtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga
     5161 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     5221 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     5281 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     5341 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta
     5401 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     5461 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgaggcgaaa tacgcgatcg
     5521 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     5581 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     5641 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     5701 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     5761 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     5821 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     5881 aaatcagcat ccatgttgga atttaatcgc ggcctggagc aagacgtttc ccgttgaata
     5941 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     6001 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt
     6061 tgaataaatc gaacttttgc tgagttgaag gatcagtcat gaccaaaatc ccttaacgtg
     6121 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
