LOCUS       sequence-235821-        7151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian cell expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7151)
  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 7151)
  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: hsa-miR-199-dendra2 PCDNA
FEATURES             Location/Qualifiers
     source          1..7151
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        119..498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        499..702
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     653..673
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     747..766
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        747..765
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1896..2588
                     /codon_start=1
                     /product="monomeric photoswitchable green-to-red 
                     fluorescent protein from Dendronephthya"
                     /label=Dendra2
                     /note="mammalian codon-optimized"
                     /translation="MNTPGINLIKEDMRVKVHMEGNVNGHAFVIEGEGKGKPYEGTQTA
                     NLTVKEGAPLPFSYDILTTAVHYGNRVFTKYPEDIPDYFKQSFPEGYSWERTMTFEDKG
                     ICTIRSDISLEGDCFFQNVRFKGTNFPPNGPVMQKKTLKWEPSTEKLHVRDGLLVGNIN
                     MALLLEGGGHYLCDFKTTYKAKKVVQLPDAHFVDHRIEILGNDSDYNKVKLYEHAVARY
                     SPLPSQVW"
     primer_bind     complement(2626..2643)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2632..2856
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2902..3330
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2989..3008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3199..3220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3339..3359)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3344..3673
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3524..3659
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3586..3605
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3740..4534
                     /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(3794..3813)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4404..4423
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4708..4829
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4745..4764)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4799..4818
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4878..4894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4878..4894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4891..4913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4902..4918
                     /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(4926..4932,4933..4950,4951..4956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4971..4992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5109..5126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5280..5868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5360..5379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6039..6830,6831..6899))
                     /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     6662..6681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6900..7004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7079..7098)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
       61 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      121 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      181 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      241 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      301 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      361 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      421 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      481 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      541 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      601 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      661 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa
      721 cccactgctt actggcttat cgaaattaat acgactcact atagggagac ccaagctggc
      781 tagcgtttaa acttaagctt gtacatttaa taaaccatcc taataacatg ttaatcatat
      841 tgaaccacat taaactctca tttgtgcatt ttacatcttt taaaaccgat ttatattcgt
      901 ttttagataa tgaagtttat taaagtgtta attcacagct ttattgcttg ttttgagaat
      961 ctgcttgcat aagtgatttt taaacttgat ctgattttcg tttgtttcga agttcttcag
     1021 ctttgaaagt cagatcttca attcacagcc atgccatagt tttaggatac agtataagta
     1081 gacgaattgg gatgcatcgt agattttatg ctgtgtttgt tatggttttt cagtggtaga
     1141 gattttcaga gtattattag ggttatagtc agtgtcaggt ttatttatcg tcagcgtgat
     1201 gggcaaaaaa cagctggcag tacgggctct ctccacagtc gtgtgcggtc ctcagggcag
     1261 tgacctacgc tccagccacc tcttgcctcc ccctcgcctg cccagtgttc agactacctg
     1321 ttcatcatgc tgcagctgaa cagtagtccg cacattggtt aggctgggct gggacacaca
     1381 cacccgccaa gccgccgtaa gatctctgcc attcgccggt gaacgaataa agcaagactt
     1441 tagccccgca caatacaccg tttcagcatc gacatcgcaa tgaaccacat cgctgaattc
     1501 acaagccaac tgctgttact ctcgcccaga aatctgcgcc aacatggaca cacccagtgg
     1561 agcaagccat ctgattttca tggtcagatt gtaaagaagc aattactctt ggacagttta
     1621 agcagaaaag ccaccaagtc aagtcactgg tcatatgatg aactcgaaga tctaataaaa
     1681 gttaatttaa gcgttcatta caataaattc aagtgaatct gttttaagct agcctttaac
     1741 gtttttaaat tccttcaaac aaattagttc ctctttgcag ttcctcattt ctcggtgagc
     1801 tggtgcttta atccctttac tttttcttcc tctagggagt gatggttggc atgggacgaa
     1861 ttaattcctg cagcccgggg gtaccggtcg ccaccatgaa caccccggga attaacctga
     1921 tcaaggagga catgcgcgtg aaggtgcaca tggagggcaa cgtgaacggc cacgccttcg
     1981 tgatcgaggg cgagggcaag ggcaagccct acgagggcac ccagaccgcc aacctgaccg
     2041 tgaaggaggg cgcccccctg cccttcagct acgacatcct gaccaccgcc gtgcactacg
     2101 gcaaccgggt gttcaccaag taccccgagg acatccccga ctacttcaag cagagcttcc
     2161 ccgagggcta cagctgggag cgcaccatga ccttcgagga caagggcatc tgcaccatcc
     2221 gcagcgacat cagcctggag ggcgactgct tcttccagaa cgtgcgcttc aagggcacca
     2281 acttcccccc caacggcccc gtgatgcaga agaaaaccct gaagtgggag cccagcaccg
     2341 agaagctgca cgtgcgcgac ggcctgctgg tgggcaacat caacatggcc ctgctgctgg
     2401 agggcggcgg ccactacctg tgcgacttca agaccaccta caaggccaag aaggtggtgc
     2461 agctgcccga cgcccacttc gtggaccacc gcatcgagat cctgggcaac gacagcgact
     2521 acaacaaggt gaagctgtac gagcacgccg tggcccgcta cagccccctg cccagccagg
     2581 tgtggtaatc tagatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2641 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2701 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2761 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2821 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2881 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2941 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3001 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3061 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3121 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3181 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3241 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3301 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3361 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3421 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3481 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3541 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3601 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3661 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3721 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3781 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3841 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3901 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3961 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4021 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4081 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4141 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4201 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4261 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4321 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4381 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4441 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4501 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4561 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4621 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4681 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4741 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4801 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4861 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4921 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4981 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5041 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5101 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5161 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5221 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5281 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5341 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5401 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5461 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5521 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5581 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5641 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5701 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5761 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5821 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5881 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5941 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6001 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6061 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6121 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6181 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6241 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6301 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6361 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6421 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6481 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6541 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6601 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6661 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6721 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6781 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6841 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6901 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6961 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7021 gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta
     7081 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     7141 tcgctgagta g
//
