LOCUS       sequence-435727-        4990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 to construct history-dependent target.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4990)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 4990)
  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-T12 GG2-ER-tagBFP-tUBQ10-GG3
FEATURES             Location/Qualifiers
     source          1..4990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(60..854)
                     /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             complement(60..854)
                     /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(171..190)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     781..800
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(1203..1505)
                     /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     complement(1305..1324)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     1628..1650
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1642..1659
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1643..1659
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1666..1685
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1666..1684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1881..2582
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNESYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(3342..3358)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3342..3358)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3355..3377)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3366..3382
                     /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(3390..3396,3397..3414,3415..3420))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3435..3456
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3573..3590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3744..4332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3824..3843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4470..4844)
                     /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"
     regulatory      4841..4850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaataatt
       61 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
      121 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
      181 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
      241 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
      301 acgagatcct cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg
      361 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
      421 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
      481 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
      541 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
      601 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
      661 gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
      721 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
      781 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
      841 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagagct tgatcccctg
      901 cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc
      961 ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag
     1021 tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt
     1081 tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga
     1141 ctggctttct acgtgaaaag gatctaggtg aagatccttt ttgataatct catgcctgac
     1201 atttatattc cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg
     1261 agatcagcca cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc
     1321 atgcgccagc tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct
     1381 gacagcagac gtgcactggc cagggggatc accatccgtc gccccggcgt gtcaataata
     1441 tcactctgta catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta
     1501 aactgccgta cgtataggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     1561 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     1621 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
     1681 tagggcgaat tgggccctct agatgcatgc tcgagcggcc gccagtgtga tggatatctg
     1741 cagaattcag gacgttgatg gtctcaacct aaacaatgaa agccttcaca ctcgctctct
     1801 tcttagctct ttccctctat ctcctgccca atccagccac tagtggcagt ggctcaggat
     1861 caggatctgg atctggctct atgagcgagc tgattaagga gaacatgcac atgaagctgt
     1921 acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc
     1981 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg
     2041 ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc
     2101 agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca
     2161 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     2221 gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc
     2281 agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc
     2341 tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg
     2401 ccaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact
     2461 atgtggacta cagactggaa agaatcaagg aggccaacaa cgagtcctac gtcgagcagc
     2521 acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt
     2581 aaatctcgtc tctgttatgc ttaagaagtt caatgtttcg tttcatgtaa aactttggtg
     2641 gtttgtgttt tggggccttg tataatccct gatgaataag tgttctacta tgtttccgtt
     2701 cctgttatct ctttctttct aatgacaagt cgaacttctt ctttatcatc gcttcgtttt
     2761 tattatctgt gcttcttttg tttaatacgc ctgcaaagtg actcgactct gtttagtgca
     2821 gttctgcgaa acttgtaaat agtccaattg ttggcctcta gtaatagatg tagcgaaagt
     2881 gttgagctgt tgggttctaa ggatggcttg aacatgttaa tcttttaggt tctgagtatg
     2941 atgaacattc gttgttgcta agaaatgcct gtaatgtccc acaaatgtag aaaatggttc
     3001 gtacctttgt ccaagcattg atatgtctga tgagaggaaa ctgcaagata ctgagcttgg
     3061 tttaacgaag gagaggcagt ttcttccttc caaagcattt catttgacaa tgccttgatc
     3121 atcttaagta gagtttctgt tgtggaaagt ttgaaacttt gaagaaacga ctctcaagta
     3181 aattgatgat cacaagtgaa agtgtatgtt gcagtgagac ctcatcacgc ctgaattcca
     3241 gcacactggc ggccgttact agtggatccg agctcggtac caagcttgat gcatagcttg
     3301 agtattctaa cgcgtcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg
     3361 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3421 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3481 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3541 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3601 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3661 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3721 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3781 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3841 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3901 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3961 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4021 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4081 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4141 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4201 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4261 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4321 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4381 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4441 ttaaattaaa aatgaagttt tagcacgtgt cagtcctgct cctcggccac gaagtgcacg
     4501 cagttgccgg ccgggtcgcg cagggcgaac tcccgccccc acggctgctc gccgatctcg
     4561 gtcatggccg gcccggaggc gtcccggaag ttcgtggaca cgacctccga ccactcggcg
     4621 tacagctcgt ccaggccgcg cacccacacc caggccaggg tgttgtccgg caccacctgg
     4681 tcctggaccg cgctgatgaa cagggtcacg tcgtcccgga ccacaccggc gaagtcgtcc
     4741 tccacgaagt cccgggagaa cccgagccgg tcggtccaga actcgaccgc tccggcgacg
     4801 tcgcgcgcgg tgagcaccgg aacggcactg gtcaacttgg ccatggtggc cctcctcacg
     4861 tgctattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4921 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat
     4981 gcggtgtgaa
//
