LOCUS       sequence-352767-        6059 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 6059)
  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 6059)
  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 V5-DEST 3XFlag-SRSF3
FEATURES             Location/Qualifiers
     source          1..6059
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(123..143)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        128..457
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      308..443
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     370..389
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             524..1318
                     /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(578..597)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1188..1207
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1494..1615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1531..1550)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1585..1604
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1664..1680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1664..1680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1677..1699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1688..1704
                     /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(1712..1718,1719..1736,1737..1742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1757..1778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1895..1912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2066..2654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2146..2165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2825..3616,3617..3685))
                     /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     3448..3467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3686..3790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3865..3884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4056..4435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4436..4639
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4590..4610
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4684..4703
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4684..4702
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4732..4756
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4760..4825
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(5320..5344)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5370..5411
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(5451..5470)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5495..5543
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      join(5745..6059,1..114)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5832..5851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     join(6042..6059,1..4)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
       61 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
      121 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
      181 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
      241 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
      301 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
      361 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
      421 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
      481 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
      541 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
      601 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
      661 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
      721 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
      781 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
      841 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
      901 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
      961 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     1021 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     1081 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     1141 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     1201 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     1261 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     1321 gggactctgg ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     1381 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     1441 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     1501 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1561 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     1621 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     1681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     1741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     1801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     1861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3781 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     3841 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     3901 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     3961 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     4021 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     4081 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     4141 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4201 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     4261 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     4321 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     4381 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     4441 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     4501 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     4561 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     4621 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     4681 aattaatacg actcactata gggagaccca agctggctag ttaagctatc aacaagtttg
     4741 tacaaaaaag caggctatgg actacaaaga ccatgacggt gattataaag atcatgatat
     4801 cgattacaag gatgacgatg acaagggcca tcgtgattcc tgtccattgg actgtaaggt
     4861 ttatgtaggc aatcttggaa acaatggcaa caagacggaa ttggaacggg cttttggcta
     4921 ctatggacca ctccgaagtg tgtgggttgc tagaaaccca cccggctttg cttttgttga
     4981 atttgaagat ccccgagatg ctgctgatgc agtccgagag ctagatggaa gaacactatg
     5041 tggctgccgt gtaagagtgg aactgtcgaa tggtgaaaaa agaagtagaa atcgtggccc
     5101 acctccctct tggggtcgtc gccctcgaga tgattatcgt aggaggagtc ctccacctcg
     5161 tcgcagatct ccaagaagga gaagcttctc tcgcagccgg agcaggtccc tttctagaga
     5221 taggagaaga gagagatcgc tgtctcggga gagaaatcac aagccgtccc gatccttctc
     5281 taggtctcgt agtcgatcta ggtcaaatga aaggaaatag cccagctttc ttgtacaaag
     5341 tggttgatct agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc
     5401 tcgattctac gcgtaccggt tagtaatgag tttaaacggg ggaggctaac tgaaacacgg
     5461 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
     5521 cacgggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     5581 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     5641 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     5701 tgccatagca gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     5761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaa
//
