LOCUS       sequence-435858-        3695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 to construct history-dependent target T2PB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3695)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 3695)
  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-T10 Circuit 1 GG4-Bxb1B-PhiC31B-GG6
FEATURES             Location/Qualifiers
     source          1..3695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(67..86)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(68..86)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(93..110)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(93..109)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(102..124)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             247..549
                     /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     428..447
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             898..1692
                     /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             898..1692
                     /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(952..971)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1562..1581
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      1892..1901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1898..2272
                     /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      2410..2998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2899..2918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3152..3169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3286..3307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3322..3327,3328..3345,3346..3352)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3360..3376
                     /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     3365..3387
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3384..3400
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3384..3400
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3543..3576
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     protein_bind    3627..3676
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
ORIGIN
        1 cctgaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc
       61 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac
      121 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
      181 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctatac
      241 gtacggcagt ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat
      301 gtacagagtg atattattga cacgccgggg cgacggatgg tgatccccct ggccagtgca
      361 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
      421 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
      481 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
      541 ggaatataaa tgtcaggcat gagattatca aaaaggatct tcacctagat ccttttcacg
      601 tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc
      661 tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg
      721 cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg
      781 ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg
      841 atctgatggc gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg
      901 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
      961 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     1021 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     1081 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     1141 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     1201 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     1261 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     1321 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     1381 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcctgacggc
     1441 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     1501 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     1561 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     1621 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     1681 gagttcttct gaattattaa cgcttacaat ttcctgatgc ggtattttct ccttacgcat
     1741 ctgtgcggta tttcacaccg catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1801 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1861 ataaatgctt caataatagc acgtgaggag ggccaccatg gccaagttga ccagtgccgt
     1921 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
     1981 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
     2041 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
     2101 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
     2161 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
     2221 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct
     2281 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     2341 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     2401 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     2461 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     2521 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     2581 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     2641 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2701 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2761 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2821 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2881 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2941 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3001 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3061 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3121 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     3181 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3241 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     3301 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     3361 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctatt
     3421 taggtgacgc gttagaatac tcaagctatg catcaagctt ggtaccgagc tcggatccac
     3481 tagtaacggc cgccagtgtg ctggaattca ggggatcgga ggtctcaagc gccgcggtgc
     3541 gggtgccagg gcgtgccctt gggctccccg ggcgcgtact ccacctctat tcagtgtttt
     3601 actcgtctca atcagaagga aagcaggccc ggatgatcct gacgacggag acggccgtcg
     3661 tcgacaagcc ggccgaaagc tgagaccaca gagtg
//
