LOCUS       sequence-393493-        5732 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NeoR nls-mNG marked rapid RMCE integration vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5732)
  AUTHORS   Nonet ML
  TITLE     Rapid generation of C. elegans single-copy transgenes combining RMCE
            and drug selection.
  JOURNAL   Genetics. 2023 Apr 20:iyad072. doi: 10.1093/genetics/iyad072.
  PUBMED    37079426
REFERENCE   2  (bases 1 to 5732)
  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: "Genetics. 
            2023 Apr 20:iyad072. doi: 10.1093/genetics/iyad072."
COMMENT     Sequence Label: pNeoG2
FEATURES             Location/Qualifiers
     source          1..5732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(21..40)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      834..1422
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1323..1342
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1576..1593
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2051..2851
                     /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="GNMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2111..2130)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2721..2740
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(4714..4734)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(4747..4780)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    4801..4834
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    4904..4937
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      4949..5404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5036..5055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5246..5267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5430..5534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5535..5603,5604..5732,1..663)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
       61 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
      121 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
      181 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
      241 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
      301 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
      361 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
      421 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
      481 tggtttattg ctgataaatc tggagccggt gagcgtggat ctcgcggtat cattgcagca
      541 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
      601 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
      661 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
      721 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
      781 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
      841 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      901 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      961 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     1021 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1081 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1141 cggtcgggct gaatgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1201 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1261 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1321 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     1381 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     1441 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     1501 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     1561 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aattcgccca atacgcaaac
     1621 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1681 ggaaagcggg atcatttttg ctttcgtcgt aaatctacac acgcgtctct tccgtgcgag
     1741 agtccaagcc agcagccaaa ttcgttgact gagtattcaa cgtttatacg ttgtcggcaa
     1801 cgagaaatag gaaaatgcat cgggaaatgt tcttttttcg attttttcca aggttttgac
     1861 aaattttacc acgaattttg ctatgttttc aattaaaaaa tatgttattc aactgtttct
     1921 atgaggaaaa taaggctttg catgtaattt tcttattcag cataattttt aattaatttg
     1981 aattttctgt cctaacgttt attttgtttt cttggttatg actgatctga aattaatttt
     2041 tgaattttaa ggtaatatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     2101 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     2161 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     2221 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     2281 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     2341 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     2401 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     2461 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     2521 ggatgatctg gacgaagaac atcaggggct cgcaccagcc gaactgttcg ccaggctcaa
     2581 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2641 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2701 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaggagc ttggcggcga
     2761 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     2821 cttctatcgc cttcttgacg agttcttctg agtccaatta ctcttcaaca tccctacatg
     2881 ctctttctcc ctgtgctccc accccctatt tttgttatta tcaaaaaact tctcttaatt
     2941 tctttgtttt ttagcttctt ttaagtcacc tctaacaatg aaattgtgta gattcaaaaa
     3001 tagaattaat tcgtaataaa aagtcgaaaa aaattgtgct ccctcccccc attaataata
     3061 attctatccc aaaatctaca caatgttctg tgtacacttc ttatgttttt tacttctgat
     3121 aaattttttt gaaacatcat agaaaaaacc gcacacaaaa taccttatca tatgttacgt
     3181 ttcagtttat gaccgcaatt tttagacctg ttcgaggaca actgggaaac ttttacaaac
     3241 ttacaacatg gttcagacaa gtttaaaaat ttaagtttga gaatcaacgg gactttttct
     3301 tttttcgttt tttttaattg tgtatgcttt gaagattttc aaatttattc tagtttcaat
     3361 aaaaacagta atataattga gtgggagttt ttttgctgtg atgtttttgg tgttatttac
     3421 agtgatcttc ttctaaatag aaaatcaatg gtttattgat caaaaatatt gagaagtgtc
     3481 gaatcaattg aagcattgaa ggacaaattt ccactatcct acaaatccga tgcgtaaaaa
     3541 ttattaaaca aagtgaaaaa aatgcatttg aagaaaaaaa acaacagaga aggtgttgat
     3601 gggcttttat tgtgacacgt tgaatgtgta tgtgttactg tgcagtggta ggtttggggg
     3661 acgtagggga agagaaacgg gtaaagagat tgaaagaaaa gagagaaacg agatggaatt
     3721 agagcagttg atctacttgg cgagcttctt ggcgttctcg gagagcttgg ttgggttggc
     3781 cttacgacga cgggaggcct tgtagagttc atccattccc atcacatctg tgaaggcctt
     3841 ttgccactct ttgaagttca gctctgtctt tgagtgcttg agttctgtct ttcggaacac
     3901 gtacattggt tgattctgaa aatttaaatc agattagtta gttagtaaaa tcatgtttaa
     3961 acttaccttg aggtagttgg cggccattgg cttagcgaat gtgtatgttg ttcgggctgt
     4021 cgatcggtat cgctttccgt ttccagtagt gtacgaccac ttgaatgtcg agatgattgt
     4081 cttatcattt gggtatgtct ttttcgatcg gcaccaatca gcagctgtga gtgagtttgt
     4141 catcactggt ccatcagctg ggaatcctgt tcccttaact tgagcctctc cctgaaaatt
     4201 taaatatgta tggttagtta gtaccgaact gtttaaactt accttgatgt gcgatccctc
     4261 gtatgtgtat cggtagttca ctgtgagcga ggctccatcc tcgaattgca ttgttcggtg
     4321 aacttggtat cccgatccat ccaccatggc agcttggaat ggtgacattc catctgaaaa
     4381 tttaaatatt actcgttagt tagtagaact cagtttaaac ttacctgggt atgggaggta
     4441 ttggtggaat ccgtatccga tgtgtggcac gagaatccat ggcgagaatt gcagatctcc
     4501 ctttgtcgac ttgaggttga gttcctcgta tccatcgttt gggtttccag ttcctgaaaa
     4561 tttaaataat cagggttagt tagtatatat atgtttaaac ttaccttgtc cgaccatatc
     4621 gaaatccact ccgttgatcg atccgaagat gtggagttcg tgtgtagctg ggagtgaagc
     4681 catgttatcc tcttctccct tcgacaccat cgacacctta cgcttcttct ttggggatcc
     4741 cattttgaag ttcctatact ttctagagaa taggaacttc accgatatcg aattcctgca
     4801 gaagttccta ttcttcaaat agtataggaa cttcccgggt accgaagagc atgcaacgcg
     4861 tggttacctg caggctcttc gccactagtt ctagagcggc cgcataactt cgtataatgt
     4921 atgctatacg aagttatgag ctccgacgac gcgccctgta gcggcgcatt aagcgcggcg
     4981 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5041 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5101 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5161 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5221 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5281 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5341 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5401 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5461 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5521 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5581 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5641 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5701 agagttttcg ccccgaagaa cgttttccaa tg
//
