LOCUS       sequence-12565-e        7642 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7642)
  AUTHORS   Trujillo RD, Yue SB, Tang Y, O'Gorman WE, Chen CZ
  TITLE     The potential functions of primary microRNAs in target recognition 
            and repression.
  JOURNAL   EMBO J. 2010 Oct 6;29(19):3272-85. doi: 10.1038/emboj.2010.208. Epub
            2010 Aug 31.
  PUBMED    20808284
REFERENCE   2  (bases 1 to 7642)
  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: 
            "10.1038/emboj.2010.208"; journalName: "EMBO J"; date: "2010-10-6- 
            6"; volume: "29"; issue: "19"; pages: "3272-85"
COMMENT     Sequence Label: MXW-PGK-let-7_2A-IRES-GFP
FEATURES             Location/Qualifiers
     source          1..7642
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             988..1404
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1334..1356
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1372..1388
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1422..1922
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     1855..1874
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     misc_feature    2543..3094
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2687..2704)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2914..2933
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3106..3108,3109..3111,3112..3825)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3151..3172)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3412..3431)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3759..3780
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3917..4432
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(4601..4617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4601..4617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4614..4636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4625..4641
                     /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(4649..4655,4656..4673,4674..4679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4694..4715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4832..4849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5003..5591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5083..5102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5762..6553,6554..6622))
                     /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     6385..6404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6623..6727)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6795..6813
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6851..6873)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6973..6992
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7186..7208
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7200..7217
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7201..7217
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7420..7439
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7641..7642,1..516)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgccggaatt agatccctac cgggtagggg aggcgctttt
     1441 cccaaggcag tctggagcat gcgctttagc ggccccgctg ggcacttggc gctacacaag
     1501 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     1561 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     1621 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     1681 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     1741 gctttggctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
     1801 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     1861 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     1921 cgacctgcac tcgagatata tgagaagcgc atcagttttt ttttcttatt ctagatgagt
     1981 agcccaccta gcagcggtcg tttttttctc ttttcgtagg aaaattttca gttacactta
     2041 tttattagaa acctagattt agagactttt agaataactt tacttcctag aacacatctc
     2101 cctttgaatt tatatgtcta atttaacaac aagtactaat ccatttttca ggcaagcagg
     2161 cgattggtgg acggtctaca ctgtggatcc ggtaaggtag taggttgtat agtttggaat
     2221 attaccaccg gtgaactatg caattttcta ccttaccgga gacagaactc ttcgaagctg
     2281 cgtcgtcttg ctctcacaac tttcttttcg ttttcttcct cctcttactt tcacctcttt
     2341 gtttttcttt attttgtgct atcaattcgg agtcgcaaga actgtattcg gtgatcccta
     2401 ttgatattct tttgtactgt attcggagat attgtttcac gaactgtatt cggagaactg
     2461 ttgtatcggt aatgtatctg gagagaataa tctaatcgta tgtactgttg agtaatggaa
     2521 ttccgccccc cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     2581 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     2641 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     2701 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     2761 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     2821 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     2881 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     2941 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     3001 acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg aaccacgggg
     3061 acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatggt gagcaagggc
     3121 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3181 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3241 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
     3301 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3361 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3421 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3481 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3541 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3601 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3661 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3721 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3781 accgccgccg ggatcactct cggcatggac gagctgtaca agtaatgaat taattaagaa
     3841 ttgcggccgc gtcgacctgc agccaagctt atcgataaaa taaaagattt tatttagtct
     3901 ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac
     3961 gccattttgc aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta
     4021 ggaacagaga gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
     4081 ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag
     4141 agaaccatca gatgtttcca gggtgcccca aggacctgaa aatgaccctg tgccttattt
     4201 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     4261 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg
     4321 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     4381 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca
     4441 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga
     4501 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg
     4561 acagcgcaga agagctgggg agaattaatt cgtaatcatg gtcatagctg tttcctgtgt
     4621 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4681 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4741 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4801 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4861 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4921 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4981 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5041 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5101 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5161 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5221 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5281 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5341 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5401 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5461 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5521 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5581 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5641 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5701 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5761 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5821 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5881 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5941 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6001 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6061 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6121 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6181 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6241 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6301 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6361 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6421 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6481 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6541 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6601 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6661 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6721 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     6781 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     6841 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     6901 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     6961 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     7021 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
     7081 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     7141 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     7201 gtaaaacgac ggccagtgcc aagctcgctc tcccttatgc gactcctgca ttaggaagca
     7261 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     7321 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     7381 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     7441 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     7501 cgattagtcc aatttgttaa agacaggata tcagtggtcc aggctctagt tttgactcaa
     7561 caatatcacc agctgaagcc tatagagtac gagccataga taaaataaaa gattttattt
     7621 agtctccaga aaaagggggg aa
//
