LOCUS       sequence-128093-        4063 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing pGK for generation of GMAP-compatible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4063)
  AUTHORS   Akama-Garren EH, Joshi NS, Tammela T, Chang GP, Wagner BL, Lee DY, 
            Rideout Iii WM, Papagiannakopoulos T, Xue W, Jacks T
  TITLE     A Modular Assembly Platform for Rapid Generation of DNA Constructs.
  JOURNAL   Sci Rep. 2016 Feb 18;6:16836. doi: 10.1038/srep16836.
  PUBMED    26887506
REFERENCE   2  (bases 1 to 4063)
  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"; journalName: "Sci Rep."; 
            date: "2016-02-18"; volume: "6:16836. doi"; pages: " 
            10.1038/srep16836"
COMMENT     Sequence Label: TOPO pGK
FEATURES             Location/Qualifiers
     source          1..4063
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        333..832
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(368..391)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     765..784
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(950..969)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(951..969)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(976..993)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(976..992)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(985..1007)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1130..1432
                     /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     1311..1330
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1781..2575
                     /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             1781..2575
                     /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(1835..1854)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2445..2464
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      2776..2785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2782..3156
                     /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      3294..3882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3783..3802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4036..4053
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcag
      301 tgtgagggag tgtaaagctg gtttattcta ccgggtaggg gaggcgcttt tcccaaggca
      361 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
      421 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
      481 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
      541 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
      601 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
      661 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
      721 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
      781 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggagacc cgacattagc
      841 gctacagctt aagcgggatc cactagtaac ggccgccagt gtgctggaat tctgcagata
      901 tccatcacac tggcggccgc tcgagcatgc atctagaggg cccaattcgc cctatagtga
      961 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1021 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1081 ggcccgcacc gatcgccctt cccaacagtt gcgcagccta tacgtacggc agtttaaggt
     1141 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     1201 tgacacgccg gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa
     1261 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     1321 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     1381 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg
     1441 catgagatta tcaaaaagga tcttcaccta gatccttttc acgtagaaag ccagtccgca
     1501 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc
     1561 aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc
     1621 ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg
     1681 gaagccctgc aaagtaaact ggatggcttt ctcgccgcca aggatctgat ggcgcagggg
     1741 atcaagctct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     1801 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     1861 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     1921 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct
     1981 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     2041 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     2101 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     2161 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     2221 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     2281 agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac
     2341 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     2401 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     2461 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     2521 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgaattat
     2581 taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2641 ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2701 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2761 tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt gctcaccgcg
     2821 cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc
     2881 gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc
     2941 caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag
     3001 ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc
     3061 atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc
     3121 aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact tcatttttaa
     3181 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     3241 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3301 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3361 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3421 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3481 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3541 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3601 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3661 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3721 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3781 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3841 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3901 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3961 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4021 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag
//
