LOCUS       sequence-435729-        4895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 to insert p35S promoter to history-dependent target.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4895)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 4895)
  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"
COMMENT     Sequence Label: L0-T15 BpiI-p35s-BpiI
FEATURES             Location/Qualifiers
     source          1..4895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(949..968)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(950..968)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(975..992)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(975..991)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(984..1006)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1129..1431
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1310..1329
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1780..2574
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1780..2574
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1834..1853)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2444..2463
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2774..2783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2780..3154
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      3292..3880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3781..3800
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4034..4051
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4168..4189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4204..4209,4210..4227,4228..4234)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4242..4258
                     /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     4247..4269
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4266..4282
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4266..4282
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        complement(4418..4763)
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     complement(4455..4475)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
ORIGIN
        1 gtcttgacgg cgagttctgt tagatcctcg atttgaatct tagactccat gcatggcctt
       61 agattcagta ggaactacct ttttagagac tccaatctct attacttgcc ttggtttatg
      121 aagcaagcct tgaatcgtcc atactggaat agtacttctg atcttgagaa atatgtcttt
      181 ctctgtgttc ttgatgcaat tagtcctgaa tcttttgact gcatctttaa ccttcttggg
      241 aaggtatttg atctcctgga gattgttact cgggtagatc gtcttgatga gacttgctgc
      301 gtaggcctct ctaaccatct gtgggtcagc attctttctg aaattgaaga ggctaacctt
      361 ctcattatca gtggtgaaca tagtgtcgtc accttcacct tcgaacttcc ttcctagatc
      421 gtaaagatag aggaaatcgt ccattgtaat ctccggggca aaggagatct cttttggggc
      481 tggatcactg ctgggccttt tggttcctag cgtgagccag tgggcttttt gctttggtgg
      541 gcttgttagg gccttagcaa agctcttggg cttgagttga gcttctcctt tggggatgaa
      601 gttcaacctg tctgtttgct gacttgttgt gtacgcgtca gctgctgctc ttgcctctgt
      661 aatagtggca aatttcttgt gtgcaactcc gggaacgccg tttgttgccg cctttgtaca
      721 accccagtca tcgtatatac cggcatgtgg accgttatac acaacgtagt agttgatatg
      781 agggtgttga atacccgatt ctgctctgag aggagcaact gtgctgttaa gctcagattt
      841 ttgtgggatt ggaattcctc cctccttgtc ttccacagag tgcctgaatt ctgcagatat
      901 ccatcacact ggcggccgct cgagcatgca tctagagggc ccaattcgcc ctatagtgag
      961 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     1021 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     1081 gcccgcaccg atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt
     1141 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     1201 gacacgccgg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa
     1261 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     1321 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     1381 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     1441 atgagattat caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag
     1501 aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca
     1561 agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg
     1621 gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg
     1681 aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga
     1741 tcaagctctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     1801 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     1861 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     1921 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta
     1981 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     2041 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     2101 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     2161 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     2221 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     2281 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc
     2341 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     2401 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     2461 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     2521 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt
     2581 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2641 cgcatcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2701 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2761 gcacgtgagg agggccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     2821 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     2881 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     2941 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     3001 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     3061 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     3121 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctaaaacttc atttttaatt
     3181 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3241 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3301 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3361 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3421 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3481 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3541 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3601 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3661 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3721 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3781 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3841 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3901 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3961 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4021 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     4081 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     4141 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     4201 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     4261 tcacacagga aacagctatg accatgatta cgccaagcta tttaggtgac gcgttagaat
     4321 actcaagcta tgcatcaagc ttggtaccga gctcggatcc actagtaacg gccgccagtg
     4381 tgctggaatt caggacgttg atgaagacaa agtagcgtgt cctctccaaa tgaaatgaac
     4441 ttccttatat agaggaaggg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt
     4501 cagtggagat gtcacatcaa tccacttgct ttgtagacgt ggttggaacc tcttcttttt
     4561 ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gagagatctt
     4621 gaatgatagc ctttccttta tcgcaatgat ggcatttgta ggagccacct tccttttcta
     4681 ctgtcctttc gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgaaa
     4741 ttatcctttg ttgaaaagtc tcaatagccc tttgatcttc tgagactgta tctttgacat
     4801 ttttggagta gaccagagtg tcgtgctcca ccatgttgac gaagattttc ttcttgtcat
     4861 tgagtcgtaa aagactctgt atgaactgtt cgcca
//
