LOCUS       sequence-78379-e        5258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contain one loxP-PGK-Neo-pA-loxP cassette, used for golden-gate 
            reaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5258)
  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 5258)
  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-Neo
FEATURES             Location/Qualifiers
     source          1..5258
                     /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    complement(382..415)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        427..926
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(462..485)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     859..878
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             940..1743
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1775..1792)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1781..2005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2030..2063)
                     /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(2145..2164)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2146..2164)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2171..2188)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2171..2187)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2180..2202)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2325..2627
                     /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     2506..2525
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2976..3770
                     /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             2976..3770
                     /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"
     regulatory      3971..3980
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3977..4351
                     /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      4489..5077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4978..4997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5231..5248
                     /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 ctcacgtgct aaagggaaca
      361 aaagctggag ctccaccgcg gataacttcg tatagcatac attatacgaa gttatcgcgc
      421 cctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
      481 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
      541 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
      601 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
      661 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
      721 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
      781 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
      841 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
      901 cctcttcctc atctccgggc ctttcggcct gcagccaata tgggatcggc cattgaacaa
      961 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     1021 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     1081 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     1141 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     1201 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     1261 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     1321 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     1381 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     1441 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     1501 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     1561 ggattcatcg actgtggccg gctgggtgtg gcggatcgct atcaggacat agcgttggct
     1621 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     1681 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     1741 tgaggggatc aattctctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     1801 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1861 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1921 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1981 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagcta taacttcgta
     2041 tagcatacat tatacgaagt tatgtccaga gacctaaggg cgaattctgc agatatccat
     2101 cacactggcg gccgctcgag catgcatcta gagggcccaa ttcgccctat agtgagtcgt
     2161 attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     2221 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     2281 gcaccgatcg cccttcccaa cagttgcgca gcctatacgt acggcagttt aaggtttaca
     2341 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca
     2401 cgccggggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct
     2461 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg
     2521 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg
     2581 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga
     2641 gattatcaaa aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac
     2701 ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg
     2761 caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt
     2821 tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc
     2881 cctgcaaagt aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa
     2941 gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3001 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3061 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3121 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3181 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3241 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3301 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3361 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3421 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     3481 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     3541 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     3601 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     3661 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     3721 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg
     3781 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3841 tacaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3901 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca
     3961 cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
     4021 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
     4081 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
     4141 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
     4201 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
     4261 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     4321 cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt tttaatttaa
     4381 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4441 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4501 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4561 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4621 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4681 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4741 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4801 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4861 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4921 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4981 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5041 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5101 acggttcctg ggcttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5161 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     5221 gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
