LOCUS       sequence-117374-        6699 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 6699)
  AUTHORS   Auyeung VC, Ulitsky I, McGeary SE, Bartel DP
  TITLE     Beyond Secondary Structure: Primary-Sequence Determinants License 
            Pri-miRNA Hairpins for Processing.
  JOURNAL   Cell. 2013 Feb 14;152(4):844-58. doi: 10.1016/j.cell.2013.01.031.
  PUBMED    23415231
REFERENCE   2  (bases 1 to 6699)
  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: 
            "10.1016/j.cell.2013.01"; journalName: "Cell"; date: "2013-02-14- 
            14"; volume: "152"; issue: "4"; pages: "844-58"
COMMENT     Sequence Label: pcDNA3.2 mir-1-1 reporter hsa-mir17~20
FEATURES             Location/Qualifiers
     source          1..6699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    911..935
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1812..1836)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2189..2230
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2270..2289)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2314..2362
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2564..2992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2651..2670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2861..2882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3001..3021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3006..3335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3186..3321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3248..3267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3402..4196
                     /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(3456..3475)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4066..4085
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4372..4493
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4409..4428)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4463..4482
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4542..4558)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4542..4558)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4555..4577)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4566..4582
                     /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(4590..4596,4597..4614,4615..4620))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4635..4656
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4773..4790)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4944..5532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5024..5043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5703..6494,6495..6563))
                     /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     6326..6345
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6564..6668)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagctatca acaagtttgt acaaaaaagc aggctcatat gccccattag ggattatgct
      961 gaatttgtat ggtttatagt tgttagagtt tgaggtgtta attctaatta tctatttcaa
     1021 atttagcagg aaaaaagaga acatcacctt gtaaaactga agattgtgac cagtcagaat
     1081 aatgtcaaag tgcttacagt gcaggtagtg atatgtgcat ctactgcagt gaaggcactt
     1141 gtagcattat ggtgacagct gcctcgggaa gccaagttgg gctttaaagt gcagggcctg
     1201 ctgatgttga gtgctttttg ttctaaggtg catctagtgc agatagtgaa gtagattagc
     1261 atctactgcc ctaagtgctc cttctggcat aagaagttat gtattcatcc aataattcaa
     1321 gccaagcaag tatataggtg ttttaatagt ttttgtttgc agtcctctgt tagttttgca
     1381 tagttgcact acaagaagaa tgtagttgtg caaatctatg caaaactgat ggtggcctgc
     1441 tatttccttc aaatgaatga tttttactaa ttttgtgtac ttttattgtg tcgatgtaga
     1501 atctgcctgg tctatctgat gtgacagctt ctgtagcact aaagtgctta tagtgcaggt
     1561 agtgtttagt tatctactgc attatgagca cttaaagtac tgctagctgt agaactccag
     1621 cttcggcctg tcgcccaatc aaactgtcct gttactgaac actgttctat ggttagtttt
     1681 gcaggtttgc atccagctgt gtgatattct gctgtgcaaa tccatgcaaa actgactgtg
     1741 gtagtgaaaa gtctgtagaa aagtaaggga aactcaaacc cctttctaca caggttggga
     1801 tcggtaagct tacccagctt tcttgtacaa agtggttgat ctagagggcc cgcagtgggg
     1861 tcagcttcta ccggggcggc gtcccggggt cttggaactg catgcagact gcctgcttgg
     1921 gaaacatact tctttatatg cccatatgga cctgctaagc tatggaatgt aaagaagtat
     1981 gtatctcagg ccgggacctc tctcgccgca ctgaggggca ctccacacca cgggggccgc
     2041 cgcggccgac agccagccag gcggctgtgg tgggggtgag gctggggcgg caggctgcct
     2101 gggcgttggt ggacggtcgt gtccccgcca gcattgttct gtcactttgc ctgtgtcagc
     2161 aggtggaagt gctcaccaga cattcgaagg taagcctatc cctaaccctc tcctcggtct
     2221 cgattctacg cgtaccggtt agtaatgagt ttaaacgggg gaggctaact gaaacacgga
     2281 aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc
     2341 acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
     2401 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
     2461 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
     2521 gccatagcag atctgcgcag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2581 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2641 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2701 cgtcaagctc taaatcgggg catcccttta gggttccgat ttagtgcttt acggcacctc
     2761 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2821 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2881 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttggggatt
     2941 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     3001 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3061 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3121 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     3181 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     3241 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     3301 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3361 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3421 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3481 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3541 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3601 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3661 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3721 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3781 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3841 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3901 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3961 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4021 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4081 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4141 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4201 gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     4261 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     4321 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     4381 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4441 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4501 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     4561 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     4621 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4681 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4741 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4801 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4861 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4921 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4981 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5041 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5101 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     5161 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5221 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5281 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5341 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5401 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5461 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5521 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5581 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5641 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5701 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5761 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5821 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5881 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5941 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6001 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6061 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6121 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6181 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6241 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6301 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6361 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6421 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6481 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6541 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6601 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6661 gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
