LOCUS       sequence-78380-e        5756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contain one loxP-PGK-Hyg-pA-loxP cassette, used for golden-gate 
            reaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5756)
  AUTHORS   Luo Y, Lin L, Bolund L, Sorensen CB
  TITLE     Efficient construction of rAAV-based gene targeting vectors by 
            Golden Gate cloning.
  JOURNAL   Biotechniques. 2014 May 1;56(5):263-8. doi: 10.2144/000114169. 
            eCollection 2014 May.
  PUBMED    24806227
REFERENCE   2  (bases 1 to 5756)
  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"; doi: "10.2144/000114169";
            journalName: "Biotechniques"; date: "2014-05-1- 1"; volume: "56"; 
            issue: "5"; pages: "263-8"
COMMENT     Sequence Label: pGolden-Hyg
FEATURES             Location/Qualifiers
     source          1..5756
                     /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"
     protein_bind    349..382
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    407..631
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     620..637
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(945..1964)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMGAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIANPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     promoter        complement(1981..2480)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2029..2048)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     2422..2445
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    2492..2525
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2643..2662)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2644..2662)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2669..2686)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2669..2685)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2678..2700)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2823..3125
                     /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     3004..3023
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             3474..4268
                     /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             3474..4268
                     /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(3528..3547)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4138..4157
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      4469..4478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4475..4849
                     /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      4987..5575
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5476..5495
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5729..5746
                     /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 ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttaggt ctctggacat aacttcgtat
      361 aatgtatgct atacgaagtt atagctggtt ctttccgcct cagaagccat agagcccacc
      421 gcatccccag catgcctgct attgtcttcc caatcctccc ccttgctgtc ctgccccacc
      481 ccacccccca gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat
      541 taggaaagga cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa
      601 acaacagatg gctggcaact agaaggcaca gtcgaggctg atcagcgagc tctagatctg
      661 cacccaatcg gcaggcacgg gcggcgatct ccaatctgcg ggatcagtca gatcacccga
      721 gtgcgtgggc atgacaatcg tgccctgggg accaacacaa tccagaaggg cctgaatcac
      781 tgcgaccggc cctcccgcga cccagccgag cgagcttagc gaactgtgga cgagaactgt
      841 gccaccaagc gtaaggccgt tctctcgcat ttgccttgct aggctcgcgc gagttgctgg
      901 ctgaggcgtt ctcgaaatca gctcttgttc ggtcggcatc tactctattc ctttgccctc
      961 ggacgagtgc tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc
     1021 cagacggccg cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg
     1081 acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag
     1141 ctctgataga gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct
     1201 gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac
     1261 ggcctccaga agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc
     1321 cagtcaatga ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc
     1381 gcgtgcacga ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga
     1441 gcctgcgcga cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg
     1501 ggattagcaa tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt
     1561 ccgaatgggc cgaacccgct cgtctggcta agatcggccg cagcgatcgc acccatggcc
     1621 tccgcgaccg gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca
     1681 ccctgtgcac ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc
     1741 acttccggaa tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag
     1801 aaaccatcgg cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg
     1861 aaagcacgag attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt
     1921 tcgatcagaa acttctcgac agacgtcgcg gtgagttcag gctttccgga tctatccatt
     1981 cgaaaggccc ggagatgagg aagaggggaa cagcgcggca gacgtgcgct tttgaagcgt
     2041 gcagaatgcc gggcctccgg aggaccttcg ggcgcccgcc ccgcccctga gcccgcccct
     2101 gagcccgccc ccggacccac cccttcccag cctctgagcc cagaaagcga aggagcaaag
     2161 ctgctattgg ccgctgcccc aaaggcctac ccgcttccat tgctcagcgg tgctgtccat
     2221 ctgcacgaga ctagtgagac gtgctacttc catttgtcac gtcctgcacg acgcgagctg
     2281 cggggcgggg gggaacttcc tgactagggg aggagtagaa ggtggcgcga aggggccacc
     2341 aaagaacgga gccggttggc gcctaccggt ggatgtggaa tgtgtgcgag gccagaggcc
     2401 acttgtgtag cgccaagtgc ccagcggggc tgctaaagcg catgctccag actgccttgg
     2461 gaaaagcgcc tcccctaccc ggtagggcgc gataacttcg tataatgtat gctatacgaa
     2521 gttatccacc gcggtggagc tccagctttt gttcccttta gcacgtgaga cctaagggcg
     2581 aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt
     2641 cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg
     2701 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     2761 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctatacgtac
     2821 ggcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     2881 agagtgatat tattgacacg ccggggcgac ggatggtgat ccccctggcc agtgcacgtc
     2941 tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct
     3001 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg
     3061 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa
     3121 tataaatgtc aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga
     3181 aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
     3241 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
     3301 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
     3361 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct
     3421 gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3481 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3541 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3601 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     3661 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3721 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3781 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3841 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3901 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3961 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     4021 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4081 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4141 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4201 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4261 tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt
     4321 gcggtatttc acaccgcata caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4381 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4441 aatgcttcaa taatagcacg tgaggagggc caccatggcc aagttgacca gtgccgttcc
     4501 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt
     4561 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt
     4621 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg
     4681 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc
     4741 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg
     4801 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac acgtgctaaa
     4861 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4921 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4981 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5041 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5101 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     5161 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5221 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5281 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5341 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5401 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5461 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5521 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5581 cagcaacgcg gcctttttac ggttcctggg cttttgctgg ccttttgctc acatgttctt
     5641 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5701 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
