LOCUS       sequence-182930-        7030 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 7030)
  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 7030)
  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-hRac2 - mut 3'UTR
FEATURES             Location/Qualifiers
     source          1..7030
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      23..611
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     512..531
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        740..1097
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        740..931
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(763..783)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      948..1083
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1167..1299
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1282..1301
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1344..1362
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1366..1375
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1372..2307
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    3123..3171
                     /note="synthetic polyadenylation signal"
     promoter        3180..3931
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      3961..3970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3967..5619
                     /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             4926..4934
                     /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    5663..5784
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5674..5693)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5728..5747
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        5917..6021
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6022..6090,6091..6882)
                     /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(6240..6259)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
       61 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
      121 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
      181 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
      241 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
      301 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
      361 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      421 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      481 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      541 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      601 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      661 ttgctcacat ggctcgacag atctgcgcag caccatggcc tgaaataacc tctgaaagag
      721 gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
      781 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
      841 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
      901 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
      961 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     1021 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     1081 gcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaagctgca
     1141 gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag
     1201 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta
     1261 ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt
     1321 acagctctta aggctagagt acttaatacg actcactata ggctagccac catggcttcc
     1381 aaggtgtacg accccgagca acgcaaacgc atgatcactg ggcctcagtg gtgggctcgc
     1441 tgcaagcaaa tgaacgtgct ggactccttc atcaactact atgattccga gaagcacgcc
     1501 gagaacgccg tgatttttct gcatggtaac gctgcctcca gctacctgtg gaggcacgtc
     1561 gtgcctcaca tcgagcccgt ggctagatgc atcatccctg atctgatcgg aatgggtaag
     1621 tccggcaaga gcgggaatgg ctcatatcgc ctcctggatc actacaagta cctcaccgct
     1681 tggttcgagc tgctgaacct tccaaagaaa atcatctttg tgggccacga ctggggggct
     1741 tgtctggcct ttcactactc ctacgagcac caagacaaga tcaaggccat cgtccatgct
     1801 gagagtgtcg tggacgtgat cgagtcctgg gacgagtggc ctgacatcga ggaggatatc
     1861 gccctgatca agagcgaaga gggcgagaaa atggtgcttg agaataactt cttcgtcgag
     1921 accatgctcc caagcaagat catgcggaaa ctggagcctg aggagttcgc tgcctacctg
     1981 gagccattca aggagaaggg cgaggttaga cggcctaccc tctcctggcc tcgcgagatc
     2041 cctctcgtta agggaggcaa gcccgacgtc gtccagattg tccgcaacta caacgcctac
     2101 cttcgggcca gcgacgatct gcctaagatg ttcatcgagt ccgaccctgg gttcttttcc
     2161 aacgctattg tcgagggagc taagaagttc cctaacaccg agttcgtgaa ggtgaagggc
     2221 ctccacttca gccaggagga cgctccagat gaaatgggta agtacatcaa gagcttcgtg
     2281 gagcgcgtgc tgaagaacga gcagtaattc taggcgatcg ctcgaggggt tgcaccccag
     2341 cgctcccacc tagatgggtc tgatcctcca ggatccccac ccaaagcctg atggcacccc
     2401 ggctggccat gctgtcccct ccctgtggcg tttcttagca gatggctgca gagctttgtt
     2461 gatggtcttt tctgtactgg aggcctcctg aggccaggaa cgtgcaaatt tgcaggtgct
     2521 gcatcccaag cccctcatgc tcctgccttc ctgagggcca gaggggagcc ccaggaccca
     2581 ttaagccacc cccgtgttcc tgccgtcagt gccaactgcc gcatgtggaa gcatctaccc
     2641 gttcactcca gtcccacccc acgcctgact cccctctgga aactgcaggc cagatggttg
     2701 ctgccacaac ttgcgtacct tcagggatgg ggctcttact ccctcctgag gccagctgct
     2761 ctaatatcga tggtcctgct tgccagagag ttcctctacc cacgtttttt gagtgtctca
     2821 gaagtgtgct cctctggcct cagttctcct cttttggaac aacataaaac aaatttaatt
     2881 ttctacgcct ctggggatat ctgctcagcc aatggaaaat ctgggttcaa ccagcccctg
     2941 ccatttctta agactttctg ctgcactcac aggatcctga gctgcactta cctgtgagag
     3001 tcttcaaact tttaaacctt gccagtcagg acttttgcta ttgcaaatag aaaacccaac
     3061 tcaacctgct taagcagaaa ataaatttat tgattcaagt ttggagagcg gccgctggcc
     3121 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaggatctaa
     3181 atgagtcttc ggacctcgcg ggggccgctt aagcggtggt tagggtttgt ctgacgcggg
     3241 gggaggggga aggaacgaaa cactctcatt cggaggcggc tcggggtttg gtcttggtgg
     3301 ccacgggcac gcagaagagc gccgcgatcc tcttaagcac ccccccgccc tccgtggagg
     3361 cgggggtttg gtcggcgggt ggtaactggc gggccgctga ctcgggcggg tcgcgcgccc
     3421 cagagtgtga ccttttcggt ctgctcgcag acccccgggc ggcgccgccg cggcggcgac
     3481 gggctcgctg ggtcctaggc tccatgggga ccgtatacgt ggacaggctc tggagcatcc
     3541 gcacgactgc ggtgatatta ccggagacct tctgcgggac gagccgggtc acgcggctga
     3601 cgcggagcgt ccgttgggcg acaaacacca ggacggggca caggtacact atcttgtcac
     3661 ccggaggcgc gagggactgc aggagcttca gggagtggcg cagctgcttc atccccgtgg
     3721 cccgttgctc gcgtttgctg gcggtgtccc cggaagaaat atatttgcat gtctttagtt
     3781 ctatgatgac acaaaccccg cccagcgtct tgtcattggc gaattcgaac acgcagatgc
     3841 agtcggggcg gcgcggtccc aggtccactt cgcatattaa ggtgacgcgt gtggcctcga
     3901 acaccgagcg accctgcagc gacccgctta aaagcttggc attccggtac tgttggtaaa
     3961 gccaccatgg ccgatgctaa gaacattaag aagggccctg ctcccttcta ccctctggag
     4021 gatggcaccg ctggcgagca gctgcacaag gccatgaaga ggtatgccct ggtgcctggc
     4081 accattgcct tcaccgatgc ccacattgag gtggacatca cctatgccga gtacttcgag
     4141 atgtctgtgc gcctggccga ggccatgaag aggtacggcc tgaacaccaa ccaccgcatc
     4201 gtggtgtgct ctgagaactc tctgcagttc ttcatgccag tgctgggcgc cctgttcatc
     4261 ggagtggccg tggcccctgc taacgacatt tacaacgagc gcgagctgct gaacagcatg
     4321 ggcatttctc agcctaccgt ggtgttcgtg tctaagaagg gcctgcagaa gatcctgaac
     4381 gtgcagaaga agctgcctat catccagaag atcatcatca tggactctaa gaccgactac
     4441 cagggcttcc agagcatgta cacattcgtg acatctcatc tgcctcctgg cttcaacgag
     4501 tacgacttcg tgccagagtc tttcgacagg gacaaaacca ttgccctgat catgaacagc
     4561 tctgggtcta ccggcctgcc taagggcgtg gccctgcctc atcgcaccgc ctgtgtgcgc
     4621 ttctctcacg cccgcgaccc tattttcggc aaccagatca tccccgacac cgctattctg
     4681 agcgtggtgc cattccacca cggcttcggc atgttcacca ccctgggcta cctgatttgc
     4741 ggctttcggg tggtgctgat gtaccgcttc gaggaggagc tgttcctgcg cagcctgcaa
     4801 gactacaaaa ttcagtctgc cctgctggtg ccaaccctgt tcagcttctt cgctaagagc
     4861 accctgatcg acaagtacga cctgtctaac ctgcacgaga ttgcctctgg cggcgcccca
     4921 ctgtctaagg aggtgggcga agccgtggcc aagcgctttc atctgccagg catccgccag
     4981 ggctacggcc tgaccgagac aaccagcgcc attctgatta ccccagaggg cgacgacaag
     5041 cctggcgccg tgggcaaggt ggtgccattc ttcgaggcca aggtggtgga cctggacacc
     5101 ggcaagaccc tgggagtgaa ccagcgcggc gagctgtgtg tgcgcggccc tatgattatg
     5161 tccggctacg tgaataaccc tgaggccaca aacgccctga tcgacaagga cggctggctg
     5221 cactctggcg acattgccta ctgggacgag gacgagcact tcttcatcgt ggaccgcctg
     5281 aagtctctga tcaagtacaa gggctaccag gtggccccag ccgagctgga gtctatcctg
     5341 ctgcagcacc ctaacatttt cgacgccgga gtggccggcc tgcccgacga cgatgccggc
     5401 gagctgcctg ccgccgtcgt cgtgctggaa cacggcaaga ccatgaccga gaaggagatc
     5461 gtggactatg tggccagcca ggtgacaacc gccaagaagc tgcgcggcgg agtggtgttc
     5521 gtggacgagg tgcccaaggg cctgaccggc aagctggacg cccgcaagat ccgcgagatc
     5581 ctgatcaagg ctaagaaagg cggcaagatc gccgtgtaat aattctagag tcggggcggc
     5641 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     5701 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     5761 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     5821 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     5881 gataaggatc caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5941 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6001 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6061 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6121 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6181 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6241 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6301 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6361 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6421 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6481 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6541 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6601 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6661 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6721 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6781 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6841 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6901 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6961 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7021 tcagaccccg
//
