LOCUS       sequence-486007-        5010 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 5010)
  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 5010)
  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: pAttBFHyg
FEATURES             Location/Qualifiers
     source          1..5010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    605..638
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1043..1067
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1070..2095
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             1070..2095
                     /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(2096..2120)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2493..2526)
                     /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    2568..2637
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     2731..2748
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2864..2885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     2941..2958
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2942..2958
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2968..2987
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2968..2986
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3135..3155)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3135..3153)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3174..3190)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3174..3190)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3187..3209)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3198..3214
                     /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)."
     rep_origin      3352..3807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3439..3458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3649..3670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3833..3937
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3938..4006,4007..4798)
                     /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"
     primer_bind     complement(4156..4175)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4969..5010,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaatg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacataact tcgtataatg tatgctatac gaagttatag gtttcccgac tggaaagcgg
      661 gatcattttt gctttcgtcg taaatctaca cacgcgtctc ttccgtgcga gagtccaagc
      721 cagcagccaa attcgttgac tgagtattca acgtttatac gttgtcggca acgagaaata
      781 ggaaaatgca tcgggaaatg ttcttttttc gattttttcc aaggttttga caaattttac
      841 cacgaatttt gctatgtttt caattaaaaa atatgttatt caactgtttc tatgaggaaa
      901 ataaggcttt gcatgtaatt ttcttattca gcataatttt taattaattt gaattttctg
      961 tcctaacgtt tattttgttt tcttggttat gactgatctg aaattaattt ttgaatttta
     1021 aggtaatatg tcaggcggtg ccgcaagttt gtacaaaaaa gcaggctcca tgaaaaagcc
     1081 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     1141 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     1201 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     1261 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     1321 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     1381 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     1441 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     1501 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     1561 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     1621 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     1681 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     1741 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     1801 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     1861 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     1921 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     1981 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     2041 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagaccca
     2101 gctttcttgt acaaagtggg tccaattact cttcaacatc cctacatgct ctttctccct
     2161 gtgctcccac cccctatttt tgttattatc aaaaaacttc tcttaatttc tttgtttttt
     2221 agcttctttt aagtcacctc taacaatgaa attgtgtaga ttcaaaaata gaattaattc
     2281 gtaataaaaa gtcgaaaaaa attgtgctcc ctccccccat taataataat tctatcccaa
     2341 aatctacaca atgttctgtg tacacttctt atgtttttta cttctgataa atttttttga
     2401 aacatcatag aaaaaaccgc acacaaaata ccttatcata tgttacgttt cagtttatga
     2461 ccgcaatttt tagacctgtt cgaggacaac tggaagttcc tatactttct agagaatagg
     2521 aacttcatca actaccgcca cctcgacccg ttcatcatga tggaccagat gggtgaggtg
     2581 gagtacgcgc ccggggagcc caagggcacg ccctggcacc cgcaccgcgg cttcatgttc
     2641 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2701 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaattc
     2761 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2821 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     2881 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgcca gtcacgacgt
     2941 tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc
     3001 gggcccccgg ttaacgggtc gacggtatcg ataagcttac cgaagagcag gcctgcacat
     3061 atgttactag tggatccatg ggctcttcgc catctagagc ggccgccacc gcggtggagc
     3121 tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     3181 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaagaaaac cctggcgtta
     3241 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     3301 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     3361 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3421 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3481 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3541 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     3601 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3661 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3721 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3781 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     3841 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3901 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3961 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4021 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4081 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4141 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4201 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4261 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4321 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4381 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4441 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4501 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4561 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4621 tattgctgat aaatctggag ccggtgagcg tggatctcgc ggtatcattg cagcactggg
     4681 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4741 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4801 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4861 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4921 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4981 ttttctgcgc gtaatctgct gcttgcaaac
//
