LOCUS       sequence-203498-        6653 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For dual luciferase report assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6653)
  AUTHORS   Hsu AY, Wang D, Gurol T, Zhou W, Zhu X, Lu HY, Deng Q
  TITLE     Overexpression of microRNA-722 fine-tunes neutrophilic inflammation 
            through inhibiting Rac2 in zebrafish.
  JOURNAL   Dis Model Mech. 2017 Sep 27. pii: dmm.030791. doi: 
            10.1242/dmm.030791.
  PUBMED    28954734
REFERENCE   2  (bases 1 to 6653)
  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: "Dis Model 
            Mech. 2017 Sep 27. pii: dmm.030791. doi: 10.1242/dmm.030791."
COMMENT     Sequence Label: pSi-check2-zRac2 - mut 3'UTR
FEATURES             Location/Qualifiers
     source          1..6653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      15..603
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     504..523
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        732..1089
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        732..923
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(755..775)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      940..1075
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1159..1291
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1274..1293
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1336..1354
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1358..1367
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1364..2299
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    2738..2786
                     /note="synthetic polyadenylation signal"
     promoter        2795..3546
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      3576..3585
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3582..5234
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /db_xref="GI:42718124"
                     /protein_id="AAS38485.1"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             4541..4549
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    5278..5399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5289..5308)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5343..5362
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        5532..5636
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5637..5705,5706..6497)
                     /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     complement(5855..5874)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
       61 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      121 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      181 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      241 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      301 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      361 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      421 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      481 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      541 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      601 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      661 atggctcgac agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
      721 ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
      781 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
      841 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      901 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
      961 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     1021 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     1081 ttttgcaaaa agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg
     1141 tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag
     1201 aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt
     1261 actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct
     1321 taaggctaga gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta
     1381 cgaccccgag caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca
     1441 aatgaacgtg ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc
     1501 cgtgattttt ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca
     1561 catcgagccc gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa
     1621 gagcgggaat ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga
     1681 gctgctgaac cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc
     1741 ctttcactac tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt
     1801 cgtggacgtg atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat
     1861 caagagcgaa gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct
     1921 cccaagcaag atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt
     1981 caaggagaag ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt
     2041 taagggaggc aagcccgacg tcgtccagat tgtccgcaac tacaacgcct accttcgggc
     2101 cagcgacgat ctgcctaaga tgttcatcga gtccgaccct gggttctttt ccaacgctat
     2161 tgtcgaggga gctaagaagt tccctaacac cgagttcgtg aaggtgaagg gcctccactt
     2221 cagccaggag gacgctccag atgaaatggg taagtacatc aagagcttcg tggagcgcgt
     2281 gctgaagaac gagcagtaat tctaggcgat cgctcgagag atacacgatt cgtcactgta
     2341 aaaaagaaaa aggtaccaga ggaatcaact ggttttctcc tataaaccaa cacaacatgc
     2401 aattgcctta cgaaagcttt tcctcaattt ttagctgaat atatcttttg aacatgccac
     2461 attgatgccg atacagtgat aatgttagta acagaatatt taatcatatt ttaactgatg
     2521 tgttatgtta tattgagact ggagtgtgtt tgtcaggtac gcagttgcat attgttttct
     2581 agcgacaaca taaacagctg aattttttat tttttgttac acataattga acccacagga
     2641 aggtattaga catgaatcat attcatgtta tacgtatgga attatttttg gcagaaaatg
     2701 cgttttttaa actgtacaac tggcggccgc tggccgcaat aaaatatctt tattttcatt
     2761 acatctgtgt gttggttttt tgtgtgagga tctaaatgag tcttcggacc tcgcgggggc
     2821 cgcttaagcg gtggttaggg tttgtctgac gcggggggag ggggaaggaa cgaaacactc
     2881 tcattcggag gcggctcggg gtttggtctt ggtggccacg ggcacgcaga agagcgccgc
     2941 gatcctctta agcacccccc cgccctccgt ggaggcgggg gtttggtcgg cgggtggtaa
     3001 ctggcgggcc gctgactcgg gcgggtcgcg cgccccagag tgtgaccttt tcggtctgct
     3061 cgcagacccc cgggcggcgc cgccgcggcg gcgacgggct cgctgggtcc taggctccat
     3121 ggggaccgta tacgtggaca ggctctggag catccgcacg actgcggtga tattaccgga
     3181 gaccttctgc gggacgagcc gggtcacgcg gctgacgcgg agcgtccgtt gggcgacaaa
     3241 caccaggacg gggcacaggt acactatctt gtcacccgga ggcgcgaggg actgcaggag
     3301 cttcagggag tggcgcagct gcttcatccc cgtggcccgt tgctcgcgtt tgctggcggt
     3361 gtccccggaa gaaatatatt tgcatgtctt tagttctatg atgacacaaa ccccgcccag
     3421 cgtcttgtca ttggcgaatt cgaacacgca gatgcagtcg gggcggcgcg gtcccaggtc
     3481 cacttcgcat attaaggtga cgcgtgtggc ctcgaacacc gagcgaccct gcagcgaccc
     3541 gcttaaaagc ttggcattcc ggtactgttg gtaaagccac catggccgat gctaagaaca
     3601 ttaagaaggg ccctgctccc ttctaccctc tggaggatgg caccgctggc gagcagctgc
     3661 acaaggccat gaagaggtat gccctggtgc ctggcaccat tgccttcacc gatgcccaca
     3721 ttgaggtgga catcacctat gccgagtact tcgagatgtc tgtgcgcctg gccgaggcca
     3781 tgaagaggta cggcctgaac accaaccacc gcatcgtggt gtgctctgag aactctctgc
     3841 agttcttcat gccagtgctg ggcgccctgt tcatcggagt ggccgtggcc cctgctaacg
     3901 acatttacaa cgagcgcgag ctgctgaaca gcatgggcat ttctcagcct accgtggtgt
     3961 tcgtgtctaa gaagggcctg cagaagatcc tgaacgtgca gaagaagctg cctatcatcc
     4021 agaagatcat catcatggac tctaagaccg actaccaggg cttccagagc atgtacacat
     4081 tcgtgacatc tcatctgcct cctggcttca acgagtacga cttcgtgcca gagtctttcg
     4141 acagggacaa aaccattgcc ctgatcatga acagctctgg gtctaccggc ctgcctaagg
     4201 gcgtggccct gcctcatcgc accgcctgtg tgcgcttctc tcacgcccgc gaccctattt
     4261 tcggcaacca gatcatcccc gacaccgcta ttctgagcgt ggtgccattc caccacggct
     4321 tcggcatgtt caccaccctg ggctacctga tttgcggctt tcgggtggtg ctgatgtacc
     4381 gcttcgagga ggagctgttc ctgcgcagcc tgcaagacta caaaattcag tctgccctgc
     4441 tggtgccaac cctgttcagc ttcttcgcta agagcaccct gatcgacaag tacgacctgt
     4501 ctaacctgca cgagattgcc tctggcggcg ccccactgtc taaggaggtg ggcgaagccg
     4561 tggccaagcg ctttcatctg ccaggcatcc gccagggcta cggcctgacc gagacaacca
     4621 gcgccattct gattacccca gagggcgacg acaagcctgg cgccgtgggc aaggtggtgc
     4681 cattcttcga ggccaaggtg gtggacctgg acaccggcaa gaccctggga gtgaaccagc
     4741 gcggcgagct gtgtgtgcgc ggccctatga ttatgtccgg ctacgtgaat aaccctgagg
     4801 ccacaaacgc cctgatcgac aaggacggct ggctgcactc tggcgacatt gcctactggg
     4861 acgaggacga gcacttcttc atcgtggacc gcctgaagtc tctgatcaag tacaagggct
     4921 accaggtggc cccagccgag ctggagtcta tcctgctgca gcaccctaac attttcgacg
     4981 ccggagtggc cggcctgccc gacgacgatg ccggcgagct gcctgccgcc gtcgtcgtgc
     5041 tggaacacgg caagaccatg accgagaagg agatcgtgga ctatgtggcc agccaggtga
     5101 caaccgccaa gaagctgcgc ggcggagtgg tgttcgtgga cgaggtgccc aagggcctga
     5161 ccggcaagct ggacgcccgc aagatccgcg agatcctgat caaggctaag aaaggcggca
     5221 agatcgccgt gtaataattc tagagtcggg gcggccggcc gcttcgagca gacatgataa
     5281 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     5341 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     5401 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt
     5461 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccaggt ggcacttttc
     5521 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5581 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5641 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5701 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5761 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5821 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5881 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5941 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6001 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6061 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6121 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6181 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6241 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6301 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6361 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6421 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6481 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6541 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6601 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aag
//
