LOCUS       sequence-182776-        7334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of miR-223 and TagRFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7334)
  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 7334)
  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-223-RFP pCDNA
FEATURES             Location/Qualifiers
     source          1..7334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        24..403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        404..607
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     558..578
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     652..671
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        652..670
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      707..716
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1967..1976
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1973..2683
                     /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(2714..2731)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2720..2944
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2990..3418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3077..3096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3287..3308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3427..3447)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3432..3761
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3612..3747
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3674..3693
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3828..4622
                     /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(3882..3901)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4492..4511
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4796..4917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4833..4852)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4887..4906
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4966..4982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4966..4982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4979..5001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4990..5006
                     /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(5014..5020,5021..5038,5039..5044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5059..5080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5197..5214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5368..5956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5448..5467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6127..6918,6919..6987))
                     /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     6750..6769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6988..7092)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7167..7186)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
       61 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      361 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      421 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      481 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      541 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      601 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
      661 tcactatagg gagacccaag ctggctagcg tttaaactta agcttcgcca ccatggatga
      721 ggaaatcgct gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg
      781 agatgatgcc cctcgtgctg ttttcccctc cattgttgga cgacccagac atcaggtaca
      841 tttaataaac catcctaata acatgttaat catattgaac cacattaaac tctcatttgt
      901 gcattttaca tcttttaaaa ccgatttata ttcgttttta gataatgaag tttattaaag
      961 tgttaattca cagctttatt gcttgttttg agaatctgct tgcataagtg atttttaaac
     1021 ttgatctgat tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat cttcaattca
     1081 cagccatgcc atagttttag gatacagtat aagtagacga attgggatgc atcgtagatt
     1141 ttatgctgtg tttgttatgg tttttcagtg gtagagattt tcagagtatt attagggtta
     1201 tagtcagtgt caggtttatt tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg
     1261 gctcaggagg aagagggagg agtaaaattg aatgaccttc ctgctgcaaa cctgcattta
     1321 atatcctttt ttcctttcat ctctcaggtc catcagcaca ccccgtctct ctcctcctga
     1381 tctagactct tctcttagag tatttgacag actgtggttg acactcgatc taaaggggtg
     1441 tcagtttgtc aaatacccca agagaggggg cacgagtctt cagaattctg caacacttcg
     1501 tatgacccat gcgatcctgt tactctctca tgttcagcct ttttgtcttg tgcaaaatgt
     1561 ggaggatgac aacattcatt agccaactgc tgttactctc gcccagaaat ctgcgccaac
     1621 atggacacac ccagtggagc aagccatctg attttcatgg tcagattgta aagaagcaat
     1681 tactcttgga cagtttaagc agaaaagcca ccaagtcaag tcactggtca tatgatgaac
     1741 tcgaagatct aataaaagtt aatttaagcg ttcattacaa taaattcaag tgaatctgtt
     1801 ttaagctagc ctttaacgtt tttaaattcc ttcaaacaaa ttagttcctc tttgcagttc
     1861 ctcatttctc ggtgagctgg tgctttaatc cctttacttt ttcttcctct agggagtgat
     1921 ggttggcatg ggacgaatta attcctgcag cccgggggta ccggtcgcca ccatggtgtc
     1981 taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
     2041 gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     2101 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     2161 taccagcttc atgtacggca gcagaacctt catcaaccac acccagggca tccccgactt
     2221 ctttaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
     2281 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     2341 caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg
     2401 ctgggaggcc aacaccgaga tgctgtaccc cgctgacggc ggcctggaag gcagaagcga
     2461 catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttcaaga ccacatacag
     2521 atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg accacagact
     2581 ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc
     2641 cagatactgc gacctcccta gcaaactggg gcacaaactt aatctagagg gcccgtttaa
     2701 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2761 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2821 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2881 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     2941 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3001 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3061 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3181 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3301 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3361 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3421 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3481 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3541 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     3601 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     3661 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     3721 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     3781 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3841 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3901 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3961 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4021 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4081 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4141 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4201 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4261 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4321 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4381 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4441 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4501 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4561 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4621 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4681 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4741 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4801 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4861 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4921 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4981 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5041 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5101 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5161 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5221 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5281 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5341 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5401 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5461 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5521 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5581 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5641 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5701 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5761 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5821 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5881 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5941 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6001 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6061 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6121 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6181 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6241 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6301 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6361 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6421 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6481 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6541 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6601 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6661 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6721 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6781 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6841 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6901 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6961 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7021 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7081 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     7141 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     7201 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     7261 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     7321 gcgctgcttc gcga
//
