LOCUS       sequence-199080-        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: [GFP][IRES][Bla]
FEATURES             Location/Qualifiers
     source          1..7585
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        37..416
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        417..620
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     571..591
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     665..684
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        665..683
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      760..769
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(766..768,769..771,772..1485)
                     /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(766..768,769..771,772..1482)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(811..832)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1072..1091)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1419..1440
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1495..2068
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1518..2070
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1662..1679)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1889..1908
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2086..2097
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2978..2995)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2984..3208
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3254..3682
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3341..3360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3551..3572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3691..3711)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3696..4025
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3876..4011
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3938..3957
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4092..4886
                     /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(4146..4165)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4756..4775
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5060..5181
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5097..5116)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5151..5170
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5230..5246)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5230..5246)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5243..5265)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5254..5270
                     /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(5278..5284,5285..5302,5303..5308))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5323..5344
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5461..5478)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5632..6220)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5712..5731)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6391..7182,7183..7251))
                     /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     7014..7033
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7252..7356)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7431..7450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
       61 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      121 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      181 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      241 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      301 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      361 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      421 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      481 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      541 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      601 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg
      661 aaattaatac gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttgg
      721 taccgagctc ggatccacta gtccagtgtg gtggaattcg ccaccatggt gagcaagggc
      781 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      841 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
      901 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
      961 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1021 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1081 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1141 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1201 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1261 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1321 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1381 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1441 accgccgccg ggatcactct cggcatggac gagctgtaca agtaagcggc cgcgcccctc
     1501 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     1561 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     1621 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     1681 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     1741 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     1801 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     1861 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     1921 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     1981 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     2041 ggttttcctt tgaaaaacac gatgataata tggccaatgc ttggaatcga gggccggatt
     2101 agcgagtttg gcagccatcc tgaaacactg gtgaaggtga aggacgcaga agatcagctg
     2161 ggcgctcgcg tggggtacat agagttggat ttgaatagcg gcaagattct ggagtcattt
     2221 agaccggagg agcggtttcc aatgatgagc acatttaaag ttctgctgtg cggggcagtg
     2281 ttgtctcgga tcgatgccgg acaggagcag ctgggccggc ggatccatta cagtcagaac
     2341 gacctcgtcg agtacagtcc tgtgactgag aagcacctga ccgatgggat gaccgtccgg
     2401 gagctttgct ctgcagctat cacaatgagc gacaatactg cggctaacct gcttctgact
     2461 acaattggcg gaccaaagga gttgacggcc ttcttgcaca acatgggcga ccacgttacc
     2521 aggctcgacc ggtgggaacc agaactgaat gaagccatcc ctaatgacga aagagatact
     2581 actacccccg tggccatggc tactaccctg agaaagctgc tcaccgggga actcctcaca
     2641 ctcgcttcca gacaacagct catcgattgg atggaagctg acaaagtggc cggaccgttg
     2701 ctgagaagcg ccctgccggc tggatggttc attgcagata agtctggggc tggggagagg
     2761 ggtagccgcg gaatcatcgc cgctcttggt cctgacggca agcctagtag gatagtggtg
     2821 atctacacca ccggcagcca ggcaaccatg gatgaaagga accggcaaat agccgaaatc
     2881 ggcgcctccc tgataaagca ctggaagctg ggcaccggca ggaggttcac cacctcctga
     2941 ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3001 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3061 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3121 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3181 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3241 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3301 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3361 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3421 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3481 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3541 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3601 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3661 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3721 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3841 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3901 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3961 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4021 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4081 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4141 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4201 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4261 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4321 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4381 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4441 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4501 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4561 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4621 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4681 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4741 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4801 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4861 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4921 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4981 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5041 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5101 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5161 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5221 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5281 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5341 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5401 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5461 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5521 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5581 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5641 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5701 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5761 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5821 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5881 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5941 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6001 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6061 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6121 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6181 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6241 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6301 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6361 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6421 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6481 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6541 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6601 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6661 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6721 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6781 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6841 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6901 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6961 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7021 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7081 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7141 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7201 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7261 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7321 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7381 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     7441 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     7501 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     7561 aatctgctta gggttaggcg ttttg
//
