LOCUS       sequence-117396-        5992 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 5992)
  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 5992)
  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 mir30-2
FEATURES             Location/Qualifiers
     source          1..5992
                     /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"
     ncRNA           1013..1027
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(1105..1129)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1482..1523
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1563..1582)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1607..1655
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1857..2285
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1944..1963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2154..2175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2294..2314)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2299..2628
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2479..2614
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2541..2560
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2695..3489
                     /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(2749..2768)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3359..3378
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3665..3786
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3702..3721)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3756..3775
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3835..3851)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3835..3851)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3848..3870)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3859..3875
                     /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(3883..3889,3890..3907,3908..3913))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3928..3949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4066..4083)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4237..4825)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4317..4336)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4996..5787,5788..5856))
                     /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     5619..5638
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5857..5961)
                     /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 aggctggtta acccaacaga aggctaaaga
      961 aggtatattg ctgtagacag tgagcgactg taaacatcct cgactggaag ctgtgaagcc
     1021 acagatgggc tttcagtcgg atgtttgcag ctgcctactg cctcggactt caaggggcta
     1081 ctttaggagc aattatcttg tttaccccag ctttcttgta caaagtggtt gatctagagg
     1141 gcccgcagtg gggtcagctt ctaccggggc ggcgtcccgg ggtcttggaa ctgcatgcag
     1201 actgcctgct tgggaaacat acttctttat atgcccatat ggacctgcta agctatggaa
     1261 tgtaaagaag tatgtatctc aggccgggac ctctctcgcc gcactgaggg gcactccaca
     1321 ccacgggggc cgccgcggcc gacagccagc caggcggctg tggtgggggt gaggctgggg
     1381 cggcaggctg cctgggcgtt ggtggacggt cgtgtccccg ccagcattgt tctgtcactt
     1441 tgcctgtgtc agcaggtgga agtgctcacc agacattcga aggtaagcct atccctaacc
     1501 ctctcctcgg tctcgattct acgcgtaccg gttagtaatg agtttaaacg ggggaggcta
     1561 actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga
     1621 cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     1681 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     1741 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     1801 ggcggcaggc cctgccatag cagatctgcg cagctggggc tctagggggt atccccacgc
     1861 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1921 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1981 cgccggcttt ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc
     2041 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2101 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2161 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2221 gattttgggg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2281 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2341 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2401 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2461 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2521 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     2581 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     2641 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     2701 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     2761 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     2821 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     2881 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     2941 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3001 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3061 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3121 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3181 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     3241 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3301 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3361 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     3421 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     3481 ttcttctgag cgggactctg gggttcgcga aatgaccgac caagcgacgc ccaacctgcc
     3541 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     3601 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     3661 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     3721 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     3781 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     3841 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3901 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3961 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4021 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4081 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4141 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4201 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4261 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4321 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4381 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     4441 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4501 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4561 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4621 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4681 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4741 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4801 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4861 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4921 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4981 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5041 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5101 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5161 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5221 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5281 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5341 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5401 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5461 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5521 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5581 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5641 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5701 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5761 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5821 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5881 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5941 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
