LOCUS       sequence-261382-        6176 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control ribozyme eGFP-sTRSV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6176)
  AUTHORS   Xiang JS, Kaplan M, Dykstra P, Hinks M, McKeague M, Smolke CD
  TITLE     Massively parallel RNA device engineering in mammalian cells with 
            RNA-Seq.
  JOURNAL   Nat Commun. 2019 Sep 23;10(1):4327. doi: 10.1038/s41467-019-12334-y.
  PUBMED    31548547
REFERENCE   2  (bases 1 to 6176)
  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 
            Commun."; date: "2019-09-23"; volume: "10(1):4327. doi"; pages: " 
            10.1038/s41467-019-12334-y"
COMMENT     Sequence Label: pCS4120
FEATURES             Location/Qualifiers
     source          1..6176
                     /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"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      924..933
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(930..932,933..935,936..1649)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(930..932,933..935,936..1646)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(975..996)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1236..1255)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1583..1604
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        1668..1717
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     primer_bind     complement(1767..1784)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1773..1997
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2043..2471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2130..2149)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2340..2361
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2480..2500)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2485..2814
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2665..2800
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2727..2746
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2881..3675
                     /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(2935..2954)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3545..3564
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3849..3970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3886..3905)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3940..3959
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4019..4035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4019..4035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4032..4054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4043..4059
                     /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(4067..4073,4074..4091,4092..4097))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4112..4133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4250..4267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4421..5009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4501..4520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5180..5971,5972..6040))
                     /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     5803..5822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6041..6145)
                     /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 cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta cccgccacca tggtgagcaa gggcgaggag ctgttcaccg
      961 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     1021 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1081 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     1141 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     1201 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     1261 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     1321 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     1381 tctatatcat ggccgacaag cagaagaatg gcatcaaggt gaacttcaag atccgccaca
     1441 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1501 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     1561 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1621 ctctcggcat ggacgagctg tacaagtaac tcgagaaaca aacaaagctg tcaccggatg
     1681 tgctttccgg tctgatgagt ccgtgaggac gaaacagcaa aaagaaaaat aaaaatctag
     1741 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     1801 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     1861 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     1921 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     1981 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2041 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2101 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2161 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2221 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2281 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2341 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2401 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2461 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     2521 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     2581 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     2641 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2701 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     2761 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     2821 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     2881 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     2941 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3001 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3061 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3121 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3181 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3241 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3301 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3361 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3421 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3481 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3541 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3601 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3661 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     3721 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     3781 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     3841 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3901 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3961 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4021 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4081 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4141 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4201 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4261 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4321 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4381 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4441 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4501 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4561 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4621 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4681 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4741 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4801 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4861 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4921 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4981 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5041 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5101 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5161 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5221 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5281 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5341 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5401 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5461 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5521 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5581 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5641 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5701 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5761 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5821 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5881 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5941 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6001 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6061 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6121 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
