LOCUS       sequence-182780-        7041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for expression of miRNA and TagRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7041)
  AUTHORS   Zhou W, Pal AS, Hsu AY, Gurol T, Zhu X, Wirbisky-Hershberger SE, 
            Freeman JL, Kasinski AL, Deng Q
  TITLE     MicroRNA-223 Suppresses the Canonical NF-kappaB Pathway in Basal 
            Keratinocytes to Dampen Neutrophilic Inflammation.
  JOURNAL   Cell Rep. 2018 Feb 13;22(7):1810-1823. doi: 
            10.1016/j.celrep.2018.01.058.
  PUBMED    29444433
REFERENCE   2  (bases 1 to 7041)
  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: 
            "10.1016/j.celrep.2018.01"; journalName: "Cell Rep"; date: 
            "2018-02-13- 13"; volume: "22"; issue: "7"; pages: "1810-1823"
COMMENT     Sequence Label: miR-vector-RFP pCDNA
FEATURES             Location/Qualifiers
     source          1..7041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..588
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     539..559
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     633..652
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        633..651
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1655..1664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1661..2371
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(2402..2419)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2408..2632
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2678..3106
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2765..2784)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2975..2996
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3115..3135)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3120..3449
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3300..3435
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3362..3381
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3516..4310
                     /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(3570..3589)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4180..4199
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4484..4605
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4521..4540)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4575..4594
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4654..4670)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4654..4670)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4667..4689)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4678..4694
                     /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(4702..4708,4709..4726,4727..4732))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4747..4768
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4885..4902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5056..5644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5136..5155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5815..6606,6607..6675))
                     /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     6438..6457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6676..6780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6855..6874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      421 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      481 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      541 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      601 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      661 gctggctagc gtttaaactt aagcttcgcc accatggatg aggaaatcgc tgccctggtc
      721 gttgacaacg gctccggtat gtgcaaagcc ggttttgctg gagatgatgc ccctcgtgct
      781 gttttcccct ccattgttgg acgacccaga catcaggtac atttaataaa ccatcctaat
      841 aacatgttaa tcatattgaa ccacattaaa ctctcatttg tgcattttac atcttttaaa
      901 accgatttat attcgttttt agataatgaa gtttattaaa gtgttaattc acagctttat
      961 tgcttgtttt gagaatctgc ttgcataagt gatttttaaa cttgatctga ttttcgtttg
     1021 tttcgaagtt cttcagcttt gaaagtcaga tcttcaattc acagccatgc catagtttta
     1081 ggatacagta taagtagacg aattgggatg catcgtagat tttatgctgt gtttgttatg
     1141 gtttttcagt ggtagagatt ttcagagtat tattagggtt atagtcagtg tcaggtttat
     1201 ttatcgtcag cgtgatgggc aaaaaacagc tggcagtacg ggctatgtct tcgtctagaa
     1261 gaagacaaag ccaactgctg ttactctcgc ccagaaatct gcgccaacat ggacacaccc
     1321 agtggagcaa gccatctgat tttcatggtc agattgtaaa gaagcaatta ctcttggaca
     1381 gtttaagcag aaaagccacc aagtcaagtc actggtcata tgatgaactc gaagatctaa
     1441 taaaagttaa tttaagcgtt cattacaata aattcaagtg aatctgtttt aagctagcct
     1501 ttaacgtttt taaattcctt caaacaaatt agttcctctt tgcagttcct catttctcgg
     1561 tgagctggtg ctttaatccc tttacttttt cttcctctag ggagtgatgg ttggcatggg
     1621 acgaattaat tcctgcagcc cgggggtacc ggtcgccacc atggtgtcta agggcgaaga
     1681 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca
     1741 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat
     1801 caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat
     1861 gtacggcagc agaaccttca tcaaccacac ccagggcatc cccgacttct ttaagcagtc
     1921 cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac
     1981 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg
     2041 ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggccaa
     2101 caccgagatg ctgtaccccg ctgacggcgg cctggaaggc agaagcgaca tggccctgaa
     2161 gctcgtgggc gggggccacc tgatctgcaa cttcaagacc acatacagat ccaagaaacc
     2221 cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac cacagactgg aaagaatcaa
     2281 ggaggccgac aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga
     2341 cctccctagc aaactggggc acaaacttaa tctagagggc ccgtttaaac ccgctgatca
     2401 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2461 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2521 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2581 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2641 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     2701 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2761 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2821 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2881 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2941 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3001 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3061 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3121 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3181 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3241 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3301 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3361 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     3421 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     3481 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3541 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3601 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3661 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     3721 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3781 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3841 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3901 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3961 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4021 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     4081 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4141 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4201 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4261 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4321 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4381 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4441 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     4501 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4561 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     4621 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4681 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     4741 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4801 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4861 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     4921 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4981 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5041 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5101 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5161 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5221 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5281 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5341 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5401 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5461 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5521 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5581 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5641 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5701 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5761 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5821 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5881 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     5941 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6001 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6061 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6121 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6181 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6241 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6301 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6361 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6421 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6481 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6541 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6601 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6661 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6721 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6781 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     6841 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     6901 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     6961 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     7021 gatgtacggg ccagatatac g
//
