LOCUS       sequence-182931-        6506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  luciferase assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6506)
  AUTHORS   Hsu AY, Wang D, Liu S, Lu J, Syahirah R, Bennin DA, Huttenlocher A, 
            Umulis DM, Wan J, Deng Q
  TITLE     Phenotypical microRNA screen reveals a noncanonical role of CDK2 in 
            regulating neutrophil migration.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Aug 26. pii: 1905221116. doi: 
            10.1073/pnas.1905221116.
  PUBMED    31451657
REFERENCE   2  (bases 1 to 6506)
  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: "Proc Natl 
            Acad Sci U S A. 2019 Aug 26. pii: 1905221116. doi: 
            10.1073/pnas.1905221116."
COMMENT     Sequence Label: miR-722 pCDNA
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        15..394
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        395..598
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     549..569
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     643..662
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        643..661
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      697..706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1877..1894)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1883..2107
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2153..2581
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2240..2259)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2450..2471
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2590..2610)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2595..2924
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2775..2910
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2837..2856
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2991..3785
                     /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(3045..3064)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3655..3674
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3959..4080
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3996..4015)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4050..4069
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4129..4145)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4129..4145)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4142..4164)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4153..4169
                     /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(4177..4183,4184..4201,4202..4207))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4222..4243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4360..4377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4531..5119)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4611..4630)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5290..6081,6082..6150))
                     /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     5913..5932
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6151..6255)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6330..6349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
       61 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      121 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      181 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      241 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      301 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      361 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      421 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      481 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      541 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
      601 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
      661 ggagacccaa gctggctagc gtttaaactt aagcttgcca ccatggatga ggaaatcgct
      721 gccctggtcg ttgacaacgg ctccggtatg tgcaaagccg gttttgctgg agatgatgcc
      781 cctcgtgctg ttttcccctc cattgttgga cgacccagac atcaggtaca tttaataaac
      841 catcctaata acatgttaat catattgaac cacattaaac tctcatttgt gcattttaca
      901 tcttttaaaa ccgatttata ttcgttttta gataatgaag tttattaaag tgttaattca
      961 cagctttatt gcttgttttg agaatctgct tgcataagtg atttttaaac ttgatctgat
     1021 tttcgtttgt ttcgaagttc ttcagctttg aaagtcagat cttcaattca cagccatgcc
     1081 atagttttag gatacagtat aagtagacga attgggatgc atcgtagatt ttatgctgtg
     1141 tttgttatgg tttttcagtg gtagagattt tcagagtatt attagggtta tagtcagtgt
     1201 caggtttatt tatcgtcagc gtgatgggca aaaaacagct ggcagtacgg gctaatcagg
     1261 actgtgttgc tgtctgtgtg gctgcgcaga tgttggggac gtccagcgga gacctgtgta
     1321 ttggaacgga gtggaatttg aaacgttttg gccaaaaatg tagccatggc aaaggggttt
     1381 tttgcagaaa cgtttcagat ttcgctccgt tctccggctt cggaaaaaac ctgctgctgc
     1441 cgagaggaag acgaagagga gccaactgct gttactctcg cccagaaatc tgcgccaaca
     1501 tggacacacc cagtggagca agccatctga ttttcatggt cagattgtaa agaagcaatt
     1561 actcttggac agtttaagca gaaaagccac caagtcaagt cactggtcat atgatgaact
     1621 cgaagatcta ataaaagtta atttaagcgt tcattacaat aaattcaagt gaatctgttt
     1681 taagctagcc tttaacgttt ttaaattcct tcaaacaaat tagttcctct ttgcagttcc
     1741 tcatttctcg gtgagctggt gctttaatcc ctttactttt tcttcctcta gggagtgatg
     1801 gttggcatgg gacgaattaa ttcctgcagc ccgggggtac cggtctctag agggcccgtt
     1861 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     1921 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     1981 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     2041 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     2101 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2161 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2221 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2281 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2341 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2401 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2461 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2521 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2581 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     2641 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     2701 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     2761 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     2821 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     2881 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     2941 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     3001 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     3061 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     3121 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     3181 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     3241 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     3301 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     3361 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3421 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3481 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     3541 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3601 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3661 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3721 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3781 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     3841 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     3901 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     3961 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4021 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4081 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4141 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4201 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4261 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4321 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4381 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4441 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4501 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4561 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4621 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4681 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4741 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4801 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4861 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4921 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4981 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5041 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5101 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5161 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5221 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5281 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5341 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5401 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5461 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5521 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5581 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5641 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5701 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5761 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5821 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5881 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5941 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6001 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6061 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6121 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6181 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6241 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6301 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     6361 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     6421 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     6481 tttgcgctgc ttcgcgatgt acgggc
//
