LOCUS       sequence-228459-        7898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Sec7-Halox2 at the endogenous locus via pop-in pop-out 
            mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7898)
  AUTHORS   Casler JC, Papanikou E, Barrero JJ, Glick BS
  TITLE     Maturation-driven transport and AP-1-dependent recycling of a 
            secretory cargo in the Golgi.
  JOURNAL   J Cell Biol. 2019 May 6;218(5):1582-1601. doi: 
            10.1083/jcb.201807195. Epub 2019 Mar 11.
  PUBMED    30858194
REFERENCE   2  (bases 1 to 7898)
  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"; doi: 
            "10.1083/jcb.201807195"; journalName: "J Cell Biol"; date: 
            "2019-05-6- 6"; volume: "218"; issue: "5"; pages: "1582-1601"
COMMENT     Sequence Label: pSec7-Halox2
FEATURES             Location/Qualifiers
     source          1..7898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     168..190
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     182..199
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     183..199
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             813..1703
                     /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             1731..2621
                     /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(4360..4376)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4360..4376)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4373..4395)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4384..4400
                     /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)."
     promoter        complement(join(4408..4414,4415..4432,4433..4438))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4453..4474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4591..4608)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4762..5350)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4842..4861)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5521..6312,6313..6381))
                     /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     6144..6163
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6382..6486)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(6616..7416)
                     /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
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQLN"
     promoter        complement(7417..7632)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     7675..7693
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7731..7753)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7853..7872
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct
       61 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      121 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      181 ttgtaaaacg acggccagtg aattctattg ttgtcaaatg tgttttacag cttttgatga
      241 tagagctgct caacgaatta ttcgagaacg aagattttgc ccactgtatc ccttacaagg
      301 aagccattag aattacaaga ttgttggaga aatcgtacga attttctcgt gactttaatg
      361 aagattatgg gttaagaaca agactagtag aggctcgtgt agttgataaa ataccaaacc
      421 tgttgaaaca agaaacgagt gctgctgcag ttctccttga cattatgttt cagttgtatc
      481 tgaacgatga tgagaagaag gctgatttaa taactcgtct gatcaccatt tgcatccagg
      541 tcgtagaggg ttatgtttct ttggatgata gaaccatgga acgtagtatc aatgcctggc
      601 gttcagtgat agttgaaata ttacaaggtt actacgaatt tgatgatgaa gactttagac
      661 tatactgtcc tgctatgtat gctctggtga ttcaaatatt agataagagc gttccaacgg
      721 agttgagaca tgctataaaa caatttctaa gcagagttgg tgaattatac ctttctacgg
      781 atccggctag cggaggaagt tcggggagtt cgatggcaga aatcggtact ggctttccat
      841 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
      901 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
      961 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
     1021 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
     1081 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
     1141 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
     1201 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
     1261 aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg
     1321 agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc
     1381 gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc
     1441 caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc
     1501 accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg
     1561 ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg
     1621 gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc
     1681 tgtcgacgct cgagatttcc ggctctagcg gaggaagttc ggggagttcg atggcagaaa
     1741 tcggtactgg ctttccattc gacccccatt atgtggaagt cctgggcgag cgcatgcact
     1801 acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt cctgcacggt aacccgacct
     1861 cctcctacgt gtggcgcaac atcatcccgc atgttgcacc gacccatcgc tgcattgctc
     1921 cagacctgat cggtatgggc aaatccgaca aaccagacct gggttatttc ttcgacgacc
     1981 acgtccgctt catggatgcc ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca
     2041 ttcacgactg gggctccgct ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca
     2101 aaggtattgc atttatggag ttcatccgcc ctatcccgac ctgggacgaa tggccagaat
     2161 ttgcccgcga gaccttccag gccttccgca ccaccgacgt cggccgcaag ctgatcatcg
     2221 atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag
     2281 tcgagatgga ccattaccgc gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc
     2341 gcttcccaaa cgagctgcca atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag
     2401 aatacatgga ctggctgcac cagtcccctg tcccgaagct gctgttctgg ggcaccccag
     2461 gcgttctgat cccaccggcc gaagccgctc gcctggccaa aagcctgcct aactgcaagg
     2521 ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga caacccggac ctgatcggca
     2581 gcgagatcgc gcgctggctg tcgacgctcg agatttccgg ctctagataa gcggccgggc
     2641 tttgttgaat ggtccagcag attaaaatat gcttagttgt agaacacaga aaaaatgaga
     2701 aggttgatat acttttactc atatttaccc tgatttaatg gcggtgatcc tttttagatt
     2761 cgtttaattc ataaacaagg tacatcatat agcaataaca aatatgtaaa ctatgtagaa
     2821 agacttatca agtttagcca tcttggtcct cattcatggg cattttatac taacttcatt
     2881 tttgccgaaa taagaaagga tgtaatctta acctaaaata tgtaagcaat taagggtgca
     2941 cgtagttact tgcgaacaaa taggaaaaga aagaaacaag cacggatact gaatgactga
     3001 gtgtattact acacgattta tctcaacgaa tttaagtgtg gaggacgatc ggctgcacgt
     3061 aggagtcaac gtttttttac tgtgttcatc acaaaaccat taagctgagt catccatatt
     3121 gcaaacaata aggtagcacc gcttccatca aaacactaga acataccgat gttttcacgg
     3181 atttattgga aacaaatagc agatgagggc gtttttactt gtaatgaagg cgaatgagaa
     3241 ggcaaggctt acaggcttgt tgtttatgcc aagcatcaat actcgctatg gacgagcgcc
     3301 atcagcgata acaacaaagt ttgcaagtgc ttgcttggat ataaaaggct tgacgcttaa
     3361 gataaatagg gccatacttg cttgctttta ctatttttgt tcctcataag cctgattgac
     3421 tacgctatta taaaccccta cattatccta taatcaactt cacttgtgta acgttgaatg
     3481 agttcaactg agtaatactc ggtcggaaat atacgctcca tctggcagga aacataggct
     3541 gcatttatat cttgcacaat tatgcccttc gtaggtgtat gttttcatgt tttccattat
     3601 gtcaatgtag atgtatcttt ggacaggcaa cttttggatc aataaagcac aatttttgag
     3661 caatttgaga tatccagtat ttgtacggga gtcccaagat tgtttagcct aaaaggttga
     3721 ggacttggat tattcttcca tcaagcgcct ccacccctcg catcctattt cttttgaagc
     3781 ataggggacg agactgtctg taatttatgg cgaagtatga caagaaagat tcaaactttg
     3841 ttttacgtat gccccaataa attctatggt agcactacta gagacgtatt ttggttttgt
     3901 aataattttc aaactgctcg ctatgatttg ttacaagaaa caaacaaaag acacgaccat
     3961 agggttcatg ttggtggttg cgaagccaca tctttaaaac attactgcaa attcagcagg
     4021 agtgagtagc aaaatcgaca aaaactagag tacgcaacat tcccggtcgt aaatttctta
     4081 tcgtggcgcc agaacattcc cgtccaatga tgagaatgtc tctaaaacaa atgcagtcac
     4141 agagattttc tgcgacttct cgagtgagaa tgttgcttat tggtgctagt ggcttgagat
     4201 cttcgtcaag caaattggaa tgcccccaga ttctcaaaca agcacggcag aaactgaaat
     4261 acggccaaaa ttatgaaccc ttttgtggga tctttgtaga aataggacct caggatagta
     4321 tgatgaagaa tgttggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4381 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4441 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4501 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4561 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4621 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4681 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4741 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4801 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4861 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4921 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4981 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5041 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5101 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5161 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5221 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5281 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5341 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5401 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5461 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5521 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5581 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5641 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5701 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5761 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5821 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5881 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5941 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6001 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6061 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6121 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6181 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6241 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6301 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6361 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6421 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6481 tccgcgcaca tttccccgaa aagtgccacc tagctttttc tttccaattt tttttttttc
     6541 gtcattataa aaatcattac gaccgagatt cccgggtaat aactgatata attaaattga
     6601 agctctaatt tgtgagttta gctggccgca tcttctcaaa tatgcttccc agcctgcttt
     6661 tctgtaacgt tcaccctcta ccttagcatc ccttcccttt gcaaatagtc ctcttccaac
     6721 aataataatg tcagatcctg tagagaccac atcatccacg gttctatact gttgacccaa
     6781 tgcgtctccc ttgtcatcta aacccacacc gggtgtcata atcaaccaat cgtaaccttc
     6841 atctcttcca cccatgtctc tttgagcaat aaagccgata acaaaatctt tgtcgctctt
     6901 cgcaatgtca acagtaccct tagtatattc tccagtagat agggagccct tgcatgacaa
     6961 ttctgctaac atcaaaaggc ctctaggttc ctttgttact tcttctgccg cctgcttcaa
     7021 accgctaaca atacctgggc ccaccacacc gtgtgcattc gtaatgtctg cccattctgc
     7081 tattctgtat acacccgcag agtactgcaa tttgactgta ttaccaatgt cagcaaattt
     7141 tctgtcttcg aagagtaaaa aattgtactt ggcggataat gcctttagcg gcttaactgt
     7201 gccctccatg gaaaaatcag tcaagatatc cacatgtgtt tttagtaaac aaattttggg
     7261 acctaatgct tcaactaact ccagtaattc cttggtggta cgaacatcca atgaagcaca
     7321 caagtttgtt tgcttttcgt gcatgatatt aaatagcttg gcagcaacag gactaggatg
     7381 agtagcagca cgttccttat atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt
     7441 ttttgttctg tgcagttggg ttaagaatac tgggcaattt catgtttctt caacactaca
     7501 tatgcgtata tataccaatc taagtctgtg ctccttcctt cgttcttcct tctgttcgga
     7561 gattaccgaa tcaaaaaaat ttcaaggaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa
     7621 tgatgaattg aaaagctcta agaaaccatt attatcatga cattaaccta taaaaatagg
     7681 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     7741 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     7801 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     7861 gagcagattg tactgagagt gcaccatatg cggtgtga
//
