LOCUS       sequence-486008-        4997 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  attB Hyg Resistant plasmid for RMI targeting an attP site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4997)
  AUTHORS   Nonet ML
  TITLE     Creation and manipulation of bipartite expression transgenes in C. 
            elegans using phiC31 recombinase.
  JOURNAL   bioRxiv 2024.03.01.583017
REFERENCE   2  (bases 1 to 4997)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.03.01.583017"
COMMENT     Sequence Label: pAttBFHyg2
FEATURES             Location/Qualifiers
     source          1..4997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     621..640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(859..963)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(989..1444)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1126..1147)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1338..1357
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    complement(1550..1583)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1616..1632
                     /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     1621..1643
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1640..1656
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1640..1656
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1675..1695
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1677..1695
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1843..1862)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1844..1862)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1872..1889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1872..1888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1945..1966
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2082..2099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2193..2262
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    2304..2337
                     /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    2710..2734
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(2735..3760)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(2735..3760)
                     /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"
     protein_bind    complement(3763..3787)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      complement(4236..4824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4316..4335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4995..4997,1..789,790..858))
                     /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 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
       61 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      121 tgctgcaatg ataccgcgag atccacgctc accggctcca gatttatcag caataaacca
      181 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      241 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      301 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      361 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      421 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      481 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      541 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      601 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      661 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      721 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      781 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      841 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      901 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      961 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     1021 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1081 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1141 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1201 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1261 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1321 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     1381 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     1441 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     1501 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaaa taacttcgta
     1561 tagcatacat tatacgaagt tatgttgggt aacgccaggg ttttcttgtg tggaattgtg
     1621 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     1681 aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg ctctagatgg
     1741 cgaagagccc atggatccac tagtaacata tgtgcaggcc tgctcttcgg taagcttatc
     1801 gataccgtcg acccgttaac cgggggcccg gtacccaatt cgccctatag tgagtcgtat
     1861 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact ggcatacgag ccggaagcat
     1921 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1981 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2041 cgcggggaga ggcggtttgc gtattgggcg aattccgctt cctcgctcac tgactcgctg
     2101 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2161 tccacagaat caggggataa cgcaggaaag aacatgaagc cgcggtgcgg gtgccagggc
     2221 gtgcccttgg gctccccggg cgcgtactcc acctcaccca tctggtccat catgatgaac
     2281 gggtcgaggt ggcggtagtt gatgaagttc ctattctcta gaaagtatag gaacttccag
     2341 ttgtcctcga acaggtctaa aaattgcggt cataaactga aacgtaacat atgataaggt
     2401 attttgtgtg cggttttttc tatgatgttt caaaaaaatt tatcagaagt aaaaaacata
     2461 agaagtgtac acagaacatt gtgtagattt tgggatagaa ttattattaa tggggggagg
     2521 gagcacaatt tttttcgact ttttattacg aattaattct atttttgaat ctacacaatt
     2581 tcattgttag aggtgactta aaagaagcta aaaaacaaag aaattaagag aagttttttg
     2641 ataataacaa aaataggggg tgggagcaca gggagaaaga gcatgtaggg atgttgaaga
     2701 gtaattggac ccactttgta caagaaagct gggtctattc ctttgccctc ggacgagtgc
     2761 tggggcgtcg gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg
     2821 cgcttctgcg ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt
     2881 cgcatcgacc ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga
     2941 gttggtcaag accaatgcgg agcatatacg cccggagccg cggcgatcct gcaagctccg
     3001 gatgcctccg ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga
     3061 agaagatgtt ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga
     3121 ccgctgttat gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga
     3181 ggtgccggac ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga
     3241 cggacgcact gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa
     3301 tcgcgcatat gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc
     3361 cgaacccgct cgtctggcta agatcggccg cagcgatcgc atccatggcc tccgcgaccg
     3421 gctgcagaac agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgcac
     3481 ggcgggagat gcaataggtc aggctctcgc tgaattcccc aatgtcaagc acttccggaa
     3541 tcgggagcgc ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg
     3601 cgcagctatt tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag
     3661 attcttcgcc ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa
     3721 acttctcgac agacgtcgcg gtgagttcag gctttttcat ggagcctgct tttttgtaca
     3781 aacttgcggc accgcctgac atattacctt aaaattcaaa aattaatttc agatcagtca
     3841 taaccaagaa aacaaaataa acgttaggac agaaaattca aattaattaa aaattatgct
     3901 gaataagaaa attacatgca aagccttatt ttcctcatag aaacagttga ataacatatt
     3961 ttttaattga aaacatagca aaattcgtgg taaaatttgt caaaaccttg gaaaaaatcg
     4021 aaaaaagaac atttcccgat gcattttcct atttctcgtt gccgacaacg tataaacgtt
     4081 gaatactcag tcaacgaatt tggctgctgg cttggactct cgcacggaag agacgcgtgt
     4141 gtagatttac gacgaaagca aaaatgatcc cgctttccgt gagcaaaagg ccagcaaaag
     4201 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4261 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4321 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4381 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4441 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4501 cccattcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4561 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4621 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4681 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4741 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4801 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4861 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4921 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4981 acttggtctg acagtta
//
