LOCUS       sequence-133460-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP coding region followed by enhanced synthetic inhibitory 
            BIC/miR-155 RNA (eSIBR) backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6251)
  AUTHORS   Fowler DK, Williams C, Gerritsen AT, Washbourne P
  TITLE     Improved knockdown from artificial microRNAs in an enhanced miR-155 
            backbone: a designer's guide to potent multi-target RNAi.
  JOURNAL   Nucleic Acids Res. 2015 Nov 17. pii: gkv1246.
  PUBMED    26582923
REFERENCE   2  (bases 1 to 6251)
  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: "Nucleic 
            Acids Res. 2015 Nov 17. pii: gkv1246."
COMMENT     Sequence Label: GFP-eSIBR
FEATURES             Location/Qualifiers
     source          1..6251
                     /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      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(901..903,904..906,907..1620)
                     /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(901..903,904..906,907..1617)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(946..967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1207..1226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1554..1575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(1803..1821)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1804..1821)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1841..1858)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1847..2071
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2117..2545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2204..2223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2414..2435
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2554..2574)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2559..2889
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2740..2875
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2802..2821
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2956..3750
                     /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(3010..3029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3620..3639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3924..4045
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3961..3980)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4015..4034
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4094..4110)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4094..4110)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4107..4129)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4118..4134
                     /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(4142..4148,4149..4166,4167..4172))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4187..4208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4325..4342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4496..5084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4576..4595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5255..6046,6047..6115))
                     /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     5878..5897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6116..6220)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttgccacc
      901 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      961 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1021 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1081 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     1141 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1201 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1261 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1321 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1381 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1441 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1501 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1561 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1621 ggatccacta gtaacggccg ccagtgtgct ggaattcaaa gtcgactgga ggcttgcttt
     1681 gggctgtatg ctgttgtctt caagatctgg aagacacagg acacaaggcc ctttatcagc
     1741 actcacatgg aacaaatggc caccgtggga ggatgacaac tcgagcctct agagggccct
     1801 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt
     1861 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1921 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1981 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga ggacaatagc
     2041 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2101 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2161 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2221 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2281 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2341 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2401 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2461 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     2521 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2581 agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2641 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2701 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     2761 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     2821 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     2881 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacagga
     2941 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     3001 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     3061 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     3121 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     3181 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     3241 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     3301 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     3361 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     3421 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     3481 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     3541 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     3601 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     3661 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     3721 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     3781 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     3841 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3901 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     3961 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4021 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4081 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4141 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4201 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4261 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4321 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4381 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4441 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4501 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4561 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4621 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4681 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4741 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4801 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4861 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4921 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4981 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5041 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5101 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5161 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5221 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5281 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5341 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5401 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5461 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5521 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5581 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5641 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5701 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5761 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5821 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5881 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5941 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6001 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6061 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6121 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6181 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6241 cacctgacgt c
//
