LOCUS       sequence-494803-        7200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert a C-terminal 2xHaloTag at APL2 by popin-popout.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7200)
  TITLE     Glick lab plasmids 2025
REFERENCE   2  (bases 1 to 7200)
  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"
COMMENT     Sequence Label: YIplac211-Apl2-HaloTagx2
FEATURES             Location/Qualifiers
     source          1..7200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1404..2294
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             2322..3212
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     complement(3698..3715)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3698..3714)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3707..3729)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3923..3942)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4042..4064
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4182..4985)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4986..5201)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5208..5226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5294..5398
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5399..5467,5468..6259)
                     /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(5617..5636)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6430..7018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6919..6938
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7172..7189
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgcctgcag gtcgactcta gtcgatcctt ccaacttgaa acaatgtact ttgttgttga
      301 cagaattaaa ggaatacgca atggaatatg aacctgagtt tgtttcaaga gctattcaag
      361 ctttgtccca gttgggtatt aaatatgcac aagaatcatt cgtcagtaaa gtcttggata
      421 ttctcctaga attgttagaa agacaagaca caataaaaga cgactgctgt atatcattat
      481 gtgatttatt aagacattgc cccggaaacg ataagatggc caaacaagtt tgtgcagtat
      541 ttaatacttg gtcaaatcca gaggtgttat tacaatcaga tattgcgaag tgcaactatg
      601 tctggttgct aggacagcat cccaacaatt tctcagattt ggagtcaaaa atcaatattt
      661 ttatagagaa ctttgttcaa gaggaagctt taacacagat gtctttacta atgacaatag
      721 tcagactaca tgctacttta acgggtagta tgctacaaag cgtcctagaa ttggccacac
      781 aacagacaca tgaattagat gtgcgagata tggctatgat gtattggaga tgcctttcga
      841 tgccaaataa tgaaagtttg gttaatgatc tatgtcaaaa taaacttcca atgatctcca
      901 atacactgga gaagttttca ccagaagtgc tagagaaact attaatggaa ttgggtacta
      961 taagttccat ttatttcaag cccgactcaa ataggagaaa aggcaaaaag tatgttcaaa
     1021 atatagttaa aggaaaacat atagaggaat tagaaagcat ggcaaaaaat gaaatttcta
     1081 gcaaggctaa cgatgatgtg ctattggatt ttgatgaaag agatgatgta acaaatacaa
     1141 atgcaggaat gctgaatact ttaacaactt taggcgattt ggatgattta tttgatttcg
     1201 gaccatctga ggatgccaca caaataaata caaatgatac caaggctgta caagggttga
     1261 aggagctgaa actaggtggc gactcaaacg gtatatcttc tggtggtaaa aacaatcccg
     1321 atgtttcggg cggcaatata gtgtcacagg atctcctcga tttattcacg gatccggcta
     1381 gcggaggaag ttcggggagt tcgatggcag aaatcggtac tggctttcca ttcgaccccc
     1441 attatgtgga agtcctgggc gagcgcatgc actacgtcga tgttggtccg cgcgatggca
     1501 cccctgtgct gttcctgcac ggtaacccga cctcctccta cgtgtggcgc aacatcatcc
     1561 cgcatgttgc accgacccat cgctgcattg ctccagacct gatcggtatg ggcaaatccg
     1621 acaaaccaga cctgggttat ttcttcgacg accacgtccg cttcatggat gccttcatcg
     1681 aagccctggg tctggaagag gtcgtcctgg tcattcacga ctggggctcc gctctgggtt
     1741 tccactgggc caagcgcaat ccagagcgcg tcaaaggtat tgcatttatg gagttcatcc
     1801 gccctatccc gacctgggac gaatggccag aatttgcccg cgagaccttc caggccttcc
     1861 gcaccaccga cgtcggccgc aagctgatca tcgatcagaa cgtttttatc gagggtacgc
     1921 tgccgatggg tgtcgtccgc ccgctgactg aagtcgagat ggaccattac cgcgagccgt
     1981 tcctgaatcc tgttgaccgc gagccactgt ggcgcttccc aaacgagctg ccaatcgccg
     2041 gtgagccagc gaacatcgtc gcgctggtcg aagaatacat ggactggctg caccagtccc
     2101 ctgtcccgaa gctgctgttc tggggcaccc caggcgttct gatcccaccg gccgaagccg
     2161 ctcgcctggc caaaagcctg cctaactgca aggctgtgga catcggcccg ggtctgaatc
     2221 tgctgcaaga agacaacccg gacctgatcg gcagcgagat cgcgcgctgg ctgtcgacgc
     2281 tcgagatttc cggctctagc ggaggaagtt cggggagttc gatggcagaa atcggtactg
     2341 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     2401 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     2461 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     2521 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     2581 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     2641 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     2701 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     2761 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     2821 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     2881 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     2941 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     3001 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     3061 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     3121 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     3181 cgcgctggct gtcgacgctc gagatttccg gctctagata agcggccgcc tataaacgtc
     3241 cgttgtagtg aacttaagta ttataccata catatatacc cttcgtatta atttttcctt
     3301 tttattgttg ggctgtgtgt atataattta tgtacaagaa tggatatatc aaaaaatcct
     3361 taaaagatgt aaatcaaaaa catcaatcaa cacagcttat tttgtagagc tttgtttttg
     3421 acataacttt tcaagcttgt tgattttttc taaaacaatt ttatcccttt caccatctaa
     3481 atatttctca tctgtttgat aaaacaggag gcacgatttc atttcatctc gttgcttatc
     3541 acaagtggga aaaactttca ttccatttgc cttagcacaa tctgccattg cttgaacaaa
     3601 atttgcacat tttttgtatg tttccgtctt caagttcttg cgagcctgtt gttcttcgcg
     3661 aggacttagc acccatatag gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc
     3721 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3781 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3841 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3901 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     3961 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     4021 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     4081 cgaaacgcgc gagacgaaag ggcgggtaat aactgatata attaaattga agctctaatt
     4141 tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat
     4201 cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc
     4261 cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca
     4321 tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg
     4381 ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata
     4441 aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct
     4501 ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc
     4561 tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg
     4621 tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat
     4681 ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
     4741 gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
     4801 acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
     4861 ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
     4921 aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
     4981 gacatgattt atcttcgttt cctgcatgtt tttgttctgt gcagttgggt taagaatact
     5041 gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
     5101 tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac
     5161 cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaagctggcc tcgtgatacg
     5221 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     5281 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5341 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5401 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5461 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5521 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5581 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5641 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5701 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5761 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5821 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5881 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5941 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     6001 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     6061 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     6121 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     6181 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6241 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6301 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6361 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6421 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6481 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6541 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6601 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6661 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6721 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6781 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6841 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6901 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6961 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7021 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7081 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7141 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
//
