LOCUS       sequence-358192-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid with 4x miR-124 target sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  AUTHORS   Nam HS, Capecchi MR
  TITLE     Additional gene targeting vector construction plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7522)
  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: 
            "Unpublished"
COMMENT     Sequence Label: miR-124
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        2420..2799
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2801..3078
                     /label=chicken beta-actin promoter
     intron          3079..4095
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4019..4041
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4103..4122
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    complement(4150..4183)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4185..4232
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4251..5054
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4314..4333)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4924..4943
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5153..5377
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    5543..5664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    5776..5897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    6009..6130
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(6136..6169)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    6355..6579
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6880..6899)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6881..6899)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6906..6923)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6906..6922)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6915..6937)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(7064..7519)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7201..7222)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7413..7432
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaagttt aaaccggcgg atcacaagca ataataacct gtagttttgc
     2221 tgcataaaac cccagatgac tacctatcct cccattttcc ttatttgccc ctattaaaaa
     2281 acttcccgac aaaaccgaaa atctgtggga agtcttgtcc ctccaatttt acacctgttc
     2341 aattcccctg caggacaacg cccacacacc aggttagcct ttaagcctgc ccagaagact
     2401 cccgcccatc ttctaggtcg acattgatta ttgactagtt attaatagta atcaattacg
     2461 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     2521 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     2581 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     2641 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     2701 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     2761 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
     2821 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
     2881 taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc rggsggggsg
     2941 gggsggggsg rggggsgggg sggggsgagg cggagaggkg cggcggcagc caatcagagc
     3001 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
     3061 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
     3121 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
     3181 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
     3241 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
     3301 ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg
     3361 cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc
     3421 gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa
     3481 ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg
     3541 ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg
     3601 gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg
     3661 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc
     3721 ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg
     3781 taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct
     3841 gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg
     3901 aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc
     3961 cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg
     4021 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc
     4081 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc
     4141 aaagaattca taacttcgta tagcatacat tatacgaagt tatagttgac aattaatcat
     4201 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atgggatcgg
     4261 ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4321 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4381 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4441 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4501 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4561 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4621 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4681 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4741 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4801 gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4861 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4921 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4981 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5041 acgagttctt ctgaggggat caattcgcta gcagatctcc ctcactaaag ggaacaaaag
     5101 ctggagctcc accgcggtgg cggccgctct agagctcgct gatcagcctc gactgtgcct
     5161 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     5221 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     5281 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     5341 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     5401 tggggctcga ctagagcttg cggaaccctt agctagaatc ataatcagcc ataccacatt
     5461 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     5521 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     5581 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5641 gtccaaactc atcaatgtat cttaactaga atcataatca gccataccac atttgtagag
     5701 gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat
     5761 gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5821 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5881 ctcatcaatg tatcttaact agaatcataa tcagccatac cacatttgta gaggttttac
     5941 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     6001 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     6061 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     6121 atgtatctta actagataac ttcgtatagc atacattata cgaagttata ggcgcgccca
     6181 tgttaattaa gatcggatcc ataatggcat tcaccgcgtg ccttaattcg aatggcattc
     6241 accgcgtgcc ttaaacgcgt tggcattcac cgcgtgcctt aaatgcattg gcattcaccg
     6301 cgtgccttaa cagatccact agagcggccg cctagagctc gctatcagcc tcgactgtgc
     6361 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     6421 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     6481 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     6541 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     6601 gctggggctc gactagagct tgcggaaccc ttaagcttaa agactggagt tgcagatcac
     6661 gagggaagag ggggaaggga ttctcccagg cccagggcgg tcctcagaag ccaggaggca
     6721 gcagagaact cccagaaagg tattgcaaca ctcccctccc ccctccggag aagggtgcgg
     6781 ccttctcccc gcctactcca ctgcagctcc cttactgata acaactcaga gcgactttgg
     6841 gagagcaagt gctcgagggg gggcccggta cccaattcgc cctatagtga gtcgtattac
     6901 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     6961 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     7021 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcaaattgt aagcgttaat
     7081 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     7141 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     7201 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     7261 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     7321 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     7381 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     7441 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     7501 gcgccgctac agggcgcgtc ag
//
