LOCUS       sequence-326617-        4841 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Single copy or array insertion by MosTI using split hygroR 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4841)
  AUTHORS   El Mouridi S, Alkhaldi F, Frokjaer-Jensen C
  TITLE     Modular safe-harbor transgene insertion (MosTI) for targeted 
            single-copy and extrachromosomal array integration in C. elegans.
  JOURNAL   G3 (Bethesda). 2022 Jul 28. pii: 6651068. doi: 
            10.1093/g3journal/jkac184.
  PUBMED    35900171
REFERENCE   2  (bases 1 to 4841)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "G3 
            (Bethesda). 2022 Jul 28. pii: 6651068. doi: 
            10.1093/g3journal/jkac184."
COMMENT     Sequence Label: pSEM262 - pDESTR4-R3 mosTI hygroR target
FEATURES             Location/Qualifiers
     source          1..4841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..18
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2..18
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(575..608)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    647..770
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(811..1116)
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(913..932)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1459..2118)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDMSQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGA"
     primer_bind     2033..2052
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2172..2178,2179..2196,2197..2202))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2227..2350)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2747..2763)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2747..2763)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2760..2782)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2935..3523)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3015..3034)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3640..3664)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3764..4555,4556..4624))
                     /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     4387..4406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4625..4729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4828..4841,1..9)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgtaaaacga cggccagtca tgcataatcc gcacgcatct ggaataagga agtgccattc
       61 cgcctgacct atgcatggaa caacaaacca gatattttga acgacgacac ccagaattag
      121 ccggatctcc taggcttcga gcttacatgc ttagaatcgg attagatcag ttgtaccaat
      181 ctcttgtgga tggtaacttt gacgacgctg cgtgggctca agggcgttgt gatgccattg
      241 tgcgttcagg ggcaggcacg gtaggacgaa ctcagattgc aagaaggtaa gttttcaaaa
      301 aattaagatt cagttatggg aagtgggagc ttacatattt tcaggctttc cctatccaaa
      361 acgctcgttc gagtgtttca gttcggtatg tcgaggcgtt accggacgga aaatatttaa
      421 aataaaatcc tcgtcatttc aggtcagctg cggtttggac tgacggatgc gtcgaggtcc
      481 ttgctgactc aggtaataga agaccttcta caagaccacg tgccaaagag taatctgttc
      541 gttgttccaa taaattgtta ttgttcccta tttgataact tcgtatagca tacattatac
      601 gaagttattg ccatagcatg gcctttgcag gattacgcca agctatcaac tttgtataga
      661 aaagttgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
      721 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gtcgacctgc
      781 agactggctg tgtataaggg agcctgacat ttatattccc cagaacatca ggttaatggc
      841 gtttttgatg tcattttcgc ggtggctgag atcagccact tcttccccga taacggatac
      901 cggcacactg gccatatcgg tggtcatcat gcgccagctt tcatccccga tatgcaccac
      961 cgggtaaagt tcacggggga ctttatctga cagcagacgt gcactggcca gggggatcac
     1021 catccgtcgc ccgggcgtgt caataatatc actctgtaca tccacaaaca gacgataacg
     1081 gctctctctt ttataggtgt aaaccttaaa ctgcatttca ccagcccctg ttctcgtcgg
     1141 caaaagagcc gttcatttca ataaaccggg cgacctcagc catcccttcc tgattttccg
     1201 ctttccagcg ttcggcacgc agacgacggg cttcattctg catggttgtg cttaccgaac
     1261 cggagatatt gacatcatat atgccttgag caactgatag ctgtcgctgt caactgtcac
     1321 tgtaatacgc tgcttcatag catacctctt tttgacatac ttcgggtata catatcagta
     1381 tatattctta taccgcaaaa atcagcgcgc aaaatacgca tactgttatc tggcttttag
     1441 taagccggat cctctagatt acgcccccgc ctgccactca tcgcagtact gttgtaattc
     1501 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     1561 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     1621 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     1681 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     1741 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     1801 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     1861 atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat gagcattcat
     1921 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     1981 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     2041 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggac atatcaacgg tggtatatcc
     2101 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgacg gatcctaact
     2161 caaaatccac acattatacg agccggaagc ataaagtgta aagcctgggg tgcctaatgc
     2221 ggccgccata gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc
     2281 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc aactttatta
     2341 tacatagttg ataattccct ctgacacatg cagctcgagc tggcgggagc tcaggaaacg
     2401 gagaatctgt gcggcaatgt cttatcattt gaaacgcgcc gtatcgatgc tgacatcatg
     2461 cggctcccgt gttagtcgag aagatgggta cgggccttta ttcatccaac actctacggc
     2521 ttcttcaaga gctagtaggg caccctgtag ttggaaaggg attatttcgt atggcgagcc
     2581 cataccgtct ttcttgcgga agcttaacac gataggaagt tggatagttt cgaacgatgg
     2641 ttccatacgg aacgctgtct caaggatgag tctgacggag tgtaactcga aggctaggtg
     2701 gaggctcagt gatgataagt ctgcgatggt ggatgcatgt gtcatggtca tagctgtttc
     2761 ctgtgtgaaa ttgttatccg ctcagagggc acaatcctat tccgcgctat ccgacaatct
     2821 ccaagacatt aggtggagtt cagttcggcg tatggcatat gtcgctggaa agaacatgtg
     2881 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2941 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3001 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3061 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3121 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3181 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3241 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3301 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3361 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3421 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3481 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3541 ttctacgggg tctgacgctc tattcaacaa agccgccgtc ccgtcaagtc agcgtaaatg
     3601 ggtagggggc ttcaaatcgt cctcgtgata ccaattcgga gcctgctttt ttgtacaaac
     3661 ttgttgataa tggcaattca aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3721 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3781 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3841 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3901 cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3961 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4021 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     4081 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4141 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4201 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4261 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4321 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4381 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4441 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4501 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4561 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4621 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4681 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4741 gaaaagtgcc agatacctga aacaaaaccc atcgtacggc caaggaagtc tccaataact
     4801 gtgatccacc acaagcgcca gggttttccc agtcacgacg t
//
