LOCUS       sequence-435861-        5099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0 pSPCH promoter, GG0 and GG1 BsaI sites for golden gate 
            assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5099)
  TITLE     Nemhauser Lab Plasmids 2024
REFERENCE   2  (bases 1 to 5099)
  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: L0I87-SPCHPromoter_GG0-1
FEATURES             Location/Qualifiers
     source          1..5099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(82..101)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(83..101)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(108..125)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(108..124)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(117..139)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             262..564
                     /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     443..462
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             913..1707
                     /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             913..1707
                     /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(967..986)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1577..1596
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      1907..1916
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1913..2287
                     /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      2425..3013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2914..2933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3167..3184
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3301..3322
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3337..3342,3343..3360,3361..3367)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3375..3391
                     /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     3380..3402
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3399..3415
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3399..3415
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 tgagaccaca gagtgcctga attctgcaga tatccatcac actggcggcc gctcgagcat
       61 gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
      121 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      181 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      241 ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga gagccgttat
      301 cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg gatggtgatc
      361 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
      421 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
      481 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
      541 aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag gatcttcacc
      601 tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
      661 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc
      721 agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa
      781 ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct
      841 ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga
      901 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
      961 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     1021 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     1081 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     1141 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     1201 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     1261 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     1321 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     1381 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     1441 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     1501 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     1561 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     1621 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     1681 tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct gatgcggtat
     1741 tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt ttcggggaaa
     1801 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1861 gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca ccatggccaa
     1921 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg
     1981 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg
     2041 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct
     2101 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc
     2161 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg
     2221 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca
     2281 ggactgacac gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2341 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2401 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2461 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2521 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     2581 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2641 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2701 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2761 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2821 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2881 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2941 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3001 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3061 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3121 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3181 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     3241 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     3301 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     3361 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     3421 ttacgccaag ctatttaggt gacgcgttag aatactcaag ctatgcatca agcttggtac
     3481 cgagctcgga tccactagta acggccgcca gtgtgctgga attcaggacg ttgatggtct
     3541 cacattcgtg attagagata tatccttctc tctctctctt cctcgttcgt tgttttgtct
     3601 tatggttgga tttttttttt aaacagcaaa ataaaagaaa gatttaggga aagaaagaga
     3661 cgccaaaaga catatcaagt tgagaaagag aaataaagag agtgaatgcc aaaaggagta
     3721 aatatgaggg actcgcattc atcgaggact aaactatcgc tatcaaattt caatttttat
     3781 tattatattt tgatagggtt atttattatg cggtttgtgc cgttggatga aatttggggg
     3841 aggatctagg tttttcgtgt ctcatttatg ttttagatat aaatatgcat ctacatatat
     3901 atatatggta cgtcttattg tatactttat attacagaga taaaggttgc atattgcaaa
     3961 gggaaagata cataaattcc ttaggaaaag atttttatgg gggtaaaatt ttgaataatc
     4021 acaagctcgg tataattgtt ttttgtacac acaaacatgt aatacacata aacacggaca
     4081 tgtacgttct ttgtatttaa tttacgtata tgttcgggtt gcgaatatgg tccgaccaat
     4141 cccggtttcc aagtatatat atatacaaaa aaacaaacaa aaaaaaaaca tttgaaatta
     4201 gtgagttcac atttatttta gcgcctcaat cgtgagacaa tcaattgaat gatgtgttgt
     4261 acattcacat tatagatcat tcattattca aaaaataaaa agatacatat ttagagactt
     4321 tttatgattg atagaccaat ttcagacatg ttatcgccag tcgatttaaa aatacagatt
     4381 ttcacttaaa gcatatactt tggcctttgt ttatgtcatg gtgtaatcta accaacattt
     4441 aagcccaaaa agaagaacaa aattcgattc cacaattctg aaagaaaatc cgttccttct
     4501 tctttcccga gccatacata caatttaatt aattcaattt agtaatgtta gttcagttca
     4561 actggatccc taacatattt cataaacacc tgatgagatg gcataagaat tcaatttctt
     4621 ataaaaccaa cccaaaatag aatatgaatt tagcaatatt agtaagcatt attaataaga
     4681 tgggatataa tgtgtatata tatatatata tatcacctct tttgtaagtt aatctttata
     4741 ttgatacagg gagcaaacga atttgttttc aagaaaacaa aagacaaggg atagatctaa
     4801 ggcaataaat agttgatgtc atactagacg tgcctcattt tgtctcatca aaatcaaatt
     4861 gcatcttgag gagccaccct tcatttgaac tttccctttc aaaagttttt gaaatatttg
     4921 tacataaata taaatattgc ttgacatttt cttttttgat aatccattca aatgttttag
     4981 aaaataaacg aaaatttaag aagatggagc caagacaaac caggtagtcg ttaggtttga
     5041 agttaatgag ttggctgtca gatattattt acctgaccat gtctttttag taacgctcc
//
