LOCUS       sequence-326153-        4900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single copy or array insertion by MosTI using split Pmlc-2 GFP 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4900)
  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 4900)
  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: "G3 
            (Bethesda). 2022 Jul 28. pii: 6651068. doi: 
            10.1093/g3journal/jkac184."
COMMENT     Sequence Label: pSEM264 - pDESTR4-R3 mosTI Pmlc-2_GFP target
FEATURES             Location/Qualifiers
     source          1..4900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(627..660)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    699..822
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(863..1168)
                     /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(965..984)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1511..2170)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDMSQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGA"
     primer_bind     2085..2104
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2224..2230,2231..2248,2249..2254))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2279..2402)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2805..2821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2805..2821)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2818..2840)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(2993..3581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3073..3092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(3698..3722)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(3822..4613,4614..4682))
                     /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     4445..4464
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4683..4787)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4886..4900,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4900,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtcat gcataatccg cacgcatctg gaataaggaa gtgccattcc
       61 gcctgaccta actataattc ccataatgtc tatatcatgg ccgacaagca aaagaatgga
      121 atcaaagtca acttcaagat aagacataat atagaggacg gctctgtcca gctggccgac
      181 cattaccagc agaacacccc gattggagat ggtccggtcc ttcttccaga caatcactat
      241 cttagtacgc aaagtgctct ctcgaaaggt aagttttcaa aaaattaaga ttcagttatg
      301 ggccagacac atcctattct tttcaggctt tccctatcca aaacgctcgt tcgagtgttt
      361 cagttcggta tgtcgaggcg ttaccggacg gaaaatattt aaaataaaat cctcgtcatt
      421 tcagatccta acgagaagcg tgaccatatg gttctcttag agtttgtaac cgccgctggc
      481 ataacacacg gtatggatga gctgtacaaa tcacgaagaa gaaaggctaa ccctactaaa
      541 ttgtcggaga acgcaaaaaa gttggcgaaa gaagttgaga actagtctgt tcgttgttcc
      601 aataaattgt tattgttccc tatttgataa cttcgtatag catacattat acgaagttat
      661 tgccatagca tggcctttgc aggattacgc caagctatca actttgtata gaaaagttga
      721 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
      781 gactacataa tactgtaaaa cacaacatat ccagtcacta tggtcgacct gcagactggc
      841 tgtgtataag ggagcctgac atttatattc cccagaacat caggttaatg gcgtttttga
      901 tgtcattttc gcggtggctg agatcagcca cttcttcccc gataacggat accggcacac
      961 tggccatatc ggtggtcatc atgcgccagc tttcatcccc gatatgcacc accgggtaaa
     1021 gttcacgggg gactttatct gacagcagac gtgcactggc cagggggatc accatccgtc
     1081 gcccgggcgt gtcaataata tcactctgta catccacaaa cagacgataa cggctctctc
     1141 ttttataggt gtaaacctta aactgcattt caccagcccc tgttctcgtc ggcaaaagag
     1201 ccgttcattt caataaaccg ggcgacctca gccatccctt cctgattttc cgctttccag
     1261 cgttcggcac gcagacgacg ggcttcattc tgcatggttg tgcttaccga accggagata
     1321 ttgacatcat atatgccttg agcaactgat agctgtcgct gtcaactgtc actgtaatac
     1381 gctgcttcat agcatacctc tttttgacat acttcgggta tacatatcag tatatattct
     1441 tataccgcaa aaatcagcgc gcaaaatacg catactgtta tctggctttt agtaagccgg
     1501 atcctctaga ttacgccccc gcctgccact catcgcagta ctgttgtaat tcattaagca
     1561 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     1621 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     1681 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     1741 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     1801 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     1861 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     1921 tcaccagctc accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg
     1981 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     2041 aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg
     2101 cctcaaaatg ttctttacga tgccattggg acatatcaac ggtggtatat ccagtgattt
     2161 ttttctccat tttagcttcc ttagctcctg aaaatctcga cggatcctaa ctcaaaatcc
     2221 acacattata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gcggccgcca
     2281 tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
     2341 atttaatata ttgatattta tatcatttta cgtttctcgt tcaactttat tatacatagt
     2401 tgataattcc ctctgacaca tgcagctcga gctggcggga gcacgtggtc aggaaacgga
     2461 gaatctgtgc ggcaatgtct tatcatttga aacgcgccgt atcgatgctg acatcatgcg
     2521 gctcccgtgt tagtcgagaa gatgggtacg ggcctttatt catccaacac tctacggctt
     2581 cttcaagagc tagtagggca ccctgtagtt ggaaagggat tatttcgtat ggcgagccca
     2641 taccgtcttt cttgcggaag cttaacacga taggaagttg gatagtttcg aacgatggtt
     2701 ccatacggaa cgctgtctca aggatgagtc tgacggagtg taactcgaag gctaggtgga
     2761 ggctcagtga tgataagtct gcgatggtgg atgcatgtgt catggtcata gctgtttcct
     2821 gtgtgaaatt gttatccgct cagagggcac aatcctattc cgcgctatcc gacaatctcc
     2881 aagacattag gtggagttca gttcggcgta tggcatatgt cgctggaaag aacatgtgag
     2941 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3001 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3061 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3121 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3181 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3241 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3301 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3361 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3421 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3481 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3541 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3601 ctacggggtc tgacgctcta ttcaacaaag ccgccgtccc gtcaagtcag cgtaaatggg
     3661 tagggggctt caaatcgtcc tcgtgatacc aattcggagc ctgctttttt gtacaaactt
     3721 gttgataatg gcaattcaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3781 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3841 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3901 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3961 cgagagccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4021 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4081 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4141 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4201 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4261 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4321 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4381 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4441 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4501 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4561 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4621 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4681 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4741 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4801 aaagtgccag atacctgaaa caaaacccat cgtacggcca aggaagtctc caataactgt
     4861 gatccaccac aagcgccagg gttttcccag tcacgacgtt
//
