LOCUS       sequence-128086-        4592 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TOPO construct expressing HygroR for generation of GMAP-compatible 
            gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4592)
  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 4592)
  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 HygroR
FEATURES             Location/Qualifiers
     source          1..4592
                     /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"
     CDS             329..1354
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             329..1354
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(1479..1498)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1480..1498)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1505..1522)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1505..1521)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1514..1536)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1659..1961
                     /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     1840..1859
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2310..3104
                     /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             2310..3104
                     /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(2364..2383)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2974..2993
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      3305..3314
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3311..3685
                     /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      3823..4411
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4312..4331
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4565..4582
                     /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 ctcggatcct
      301 aactcgaacg ctagctgtgc gatcgtttat gaaaaagcct gaactcaccg cgacgtctgt
      361 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
      421 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
      481 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
      541 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
      601 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
      661 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
      721 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
      781 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
      841 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
      901 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
      961 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     1021 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     1081 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     1141 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     1201 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     1261 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     1321 acgccccagc actcgtccga gggcaaagga ataaaaacgt tgttgtttgg ggttgaatta
     1381 ctctggatcc actagtaacg gccgccagtg tgctggaatt ctgcagatat ccatcacact
     1441 ggcggccgct cgagcatgca tctagagggc ccaattcgcc ctatagtgag tcgtattaca
     1501 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     1561 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     1621 atcgcccttc ccaacagttg cgcagcctat acgtacggca gtttaaggtt tacacctata
     1681 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgccgg
     1741 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     1801 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1861 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     1921 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc atgagattat
     1981 caaaaaggat cttcacctag atccttttca cgtagaaagc cagtccgcag aaacggtgct
     2041 gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga
     2101 gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga
     2161 cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca
     2221 aagtaaactg gatggctttc tcgccgccaa ggatctgatg gcgcagggga tcaagctctg
     2281 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2341 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2401 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2461 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     2521 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     2581 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     2641 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     2701 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     2761 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     2821 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     2881 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     2941 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     3001 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     3061 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgaattatt aacgcttaca
     3121 atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacagg
     3181 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3241 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat agcacgtgag
     3301 gagggccacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
     3361 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
     3421 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
     3481 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
     3541 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
     3601 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
     3661 cttcgtggcc gaggagcagg actgacacgt gctaaaactt catttttaat ttaaaaggat
     3721 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3781 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3841 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3901 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3961 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4021 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4081 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4141 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4201 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4261 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4321 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4381 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4441 cctgggcttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4501 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4561 gcgcagcgag tcagtgagcg aggaagcgga ag
//
