LOCUS       sequence-197876-        7585 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for our translation quantification assay (Fig. 5). Double 
            reporter vector with internal ribosome entry site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7585)
  AUTHORS   Adamala KP, Martin-Alarcon DA, Boyden ES
  TITLE     Programmable RNA-binding protein composed of repeats of a single 
            modular unit.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 May 10;113(19):E2579-88. doi: 
            10.1073/pnas.1519368113. Epub 2016 Apr 26.
  PUBMED    27118836
REFERENCE   2  (bases 1 to 7585)
  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"; doi: 
            "10.1073/pnas.1519368113"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-05-10- 10"; volume: "113"; issue: "19"; pages: 
            "E2579-88"
COMMENT     Sequence Label: [Bla][IRES][GFP]
FEATURES             Location/Qualifiers
     source          1..7585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        16..395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        396..599
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     550..570
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     644..663
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        644..662
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             754..765
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    1618..2191
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1641..2193
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1785..1802)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2012..2031
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2200..2202,2203..2205,2206..2919)
                     /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(2200..2202,2203..2205,2206..2916)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2245..2266)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2506..2525)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2853..2874
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2957..2974)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2963..3187
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3233..3661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3320..3339)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3530..3551
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3670..3690)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3675..4004
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3855..3990
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3917..3936
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4071..4865
                     /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(4125..4144)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4735..4754
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5039..5160
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5076..5095)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5130..5149
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5209..5225)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5209..5225)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5222..5244)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5233..5249
                     /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(5257..5263,5264..5281,5282..5287))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5302..5323
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5440..5457)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5611..6199)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5691..5710)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6370..7161,7162..7230))
                     /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     6993..7012
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7231..7335)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7410..7429)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
       61 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
      121 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
      181 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
      241 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
      301 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
      361 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
      421 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
      481 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
      541 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
      601 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
      661 gggagaccca agctggctag cgtttaaact taagcttggt accgagctcg gatccactag
      721 tccagtgtgg tggaattcgc caccatgctt ggaatcgagg gccggattag cgagtttggc
      781 agccatcctg aaacactggt gaaggtgaag gacgcagaag atcagctggg cgctcgcgtg
      841 gggtacatag agttggattt gaatagcggc aagattctgg agtcatttag accggaggag
      901 cggtttccaa tgatgagcac atttaaagtt ctgctgtgcg gggcagtgtt gtctcggatc
      961 gatgccggac aggagcagct gggccggcgg atccattaca gtcagaacga cctcgtcgag
     1021 tacagtcctg tgactgagaa gcacctgacc gatgggatga ccgtccggga gctttgctct
     1081 gcagctatca caatgagcga caatactgcg gctaacctgc ttctgactac aattggcgga
     1141 ccaaaggagt tgacggcctt cttgcacaac atgggcgacc acgttaccag gctcgaccgg
     1201 tgggaaccag aactgaatga agccatccct aatgacgaaa gagatactac tacccccgtg
     1261 gccatggcta ctaccctgag aaagctgctc accggggaac tcctcacact cgcttccaga
     1321 caacagctca tcgattggat ggaagctgac aaagtggccg gaccgttgct gagaagcgcc
     1381 ctgccggctg gatggttcat tgcagataag tctggggctg gggagagggg tagccgcgga
     1441 atcatcgccg ctcttggtcc tgacggcaag cctagtagga tagtggtgat ctacaccacc
     1501 ggcagccagg caaccatgga tgaaaggaac cggcaaatag ccgaaatcgg cgcctccctg
     1561 ataaagcact ggaagctggg caccggcagg aggttcacca cctcctgagc ggccgcgccc
     1621 ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc
     1681 gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa
     1741 acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat
     1801 gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac
     1861 aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg
     1921 cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt
     1981 tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg
     2041 gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac
     2101 atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga
     2161 cgtggttttc ctttgaaaaa cacgatgata atatggccaa tggtgagcaa gggcgaggag
     2221 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2281 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     2341 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2401 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2461 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2521 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2581 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2641 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2701 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2761 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2821 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2881 gccgggatca ctctcggcat ggacgagctg tacaagtaac tcgagtctag agggcccgtt
     2941 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3001 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3061 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3121 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3181 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     3241 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtggc cgctacactt
     3301 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3361 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3421 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3481 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3541 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3601 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3661 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3721 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3781 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3841 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3901 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     3961 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     4021 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4081 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4141 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4201 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4261 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4321 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4381 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4441 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4501 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4561 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     4621 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     4681 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     4741 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     4801 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     4861 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     4921 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4981 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     5041 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5101 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5161 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5221 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5281 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5341 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5401 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5461 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5521 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5581 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5641 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5701 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5761 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5821 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5881 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5941 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6001 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6061 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6121 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6181 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6241 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6301 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6361 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6421 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6481 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6541 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6601 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6661 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6721 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6781 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6841 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6901 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6961 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7021 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7081 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7141 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7201 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7261 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7321 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     7381 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     7441 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     7501 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     7561 tttgcgctgc ttcgcgatgt acggg
//
