LOCUS       sequence-449638-        6635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of FAM177A1-SNAPTag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6635)
  AUTHORS   Ugur B, Schueder F, Shin J, Hanna MG, Wu Y, Leonzino M, Su M, McAdow
            AR, Wilson C, Postlethwait J, Solnica-Krezel L, Bewersdorf J, De 
            Camilli P
  TITLE     VPS13B is localized at the cis-trans Golgi complex interface and is 
            a functional partner of FAM177A1.
  JOURNAL   bioRxiv [Preprint]. 2023 Dec 18:2023.12.18.572081. doi: 
            10.1101/2023.12.18.572081.
  PUBMED    38187698
REFERENCE   2  (bases 1 to 6635)
  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: "bioRxiv 
            [Preprint]. 2023 Dec 18:2023.12.18.572081. doi: 
            10.1101/2023.12.18.572081."
COMMENT     Sequence Label: FAM177A1-SNAP
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     227..247
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(256..684)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(366..387)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     578..597
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    730..954
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     943..960
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(980..1525)
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     regulatory      2242..2251
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2304..2323)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2305..2323)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2368..2571)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2397..2417)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2572..2951
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3123..3142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3217..3321
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3322..3390,3391..4182)
                     /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(3540..3559)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4353..4941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4842..4861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5095..5112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5229..5250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5265..5270,5271..5288,5289..5295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5303..5319
                     /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     5308..5330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5327..5343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5327..5343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5392..5513
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5403..5422)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5457..5476
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5687..6481)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5798..5817)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6408..6427
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6548..6635,1..242))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6562..6635,1..62)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6616..6635)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg
       61 atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata
      121 cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc
      181 atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac
      241 acattccaca gaattaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
      301 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
      361 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
      421 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
      481 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
      541 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
      601 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
      661 ttaatgcgcc gctacagggc gcgtggggat accccctaga gccccagctg gttctttccg
      721 cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct
      781 cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat
      841 gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca
      901 aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg
      961 ctgatcagcg ggtttatcaa cccagcccag gcttgcccag tctgtggccc tcgtgggcca
     1021 gcagccactc tttcacggcg agcccgccct cgtagccccc cacgtccagg tcgccctgca
     1081 ccacccggtg gcaggggatc agaatgggca cgggatttcc gctcagggcg gttttcacgg
     1141 cggcggtggc ggcgggattg ccggccaggg cggccaggtg gctgtagctg atgacctctc
     1201 cgaacttcac cactttcagc agtttccaca gcacctggcg ggtaaagctc tcctgctgga
     1261 acactgggtg gtgcagggct ggcacaggga actcctcgat ggcctcaggc tggtgaaagt
     1321 aggcgttgag ccaggcggtg gcctgcatca gtggctctgg tccgcccagc acggcggctg
     1381 gggcaggcac ttccacggcg tcggcggcag atgttccttt gcccaggaag atgatacggt
     1441 gcaggccctg ttcgcaccca gacagttcca gcttgcccag agggctatcc agggtggtgc
     1501 gcttcatttc gcagtctttg tccattccac ttcctcctgg tgggacagag actggatttt
     1561 gcttgctttc cataattact tcactgtctc cctccatttc aaaattgaca ttcacaaatg
     1621 agctggatat cactgtctct ggttgatctg tctgaacaat ggaatcagtc tgcaatttat
     1681 tctgttgata ttgtttttct gcttcttcag acatcctgtt ttcttcttct tcttcttctt
     1741 cctccttctt catccgataa tattcatcaa tggcatattg gtactttggg gtgctgatac
     1801 ccaaaacaga tgcaatcttc tctccaagga agtcacacac tgagagagtt gatgtagcag
     1861 cccgaagcat gtaaaaccat aagtagggac cccaggtaag ttttgtcgga tcaacagtag
     1921 gcaaaacatc tttcttctcc aggccatcaa cttcgtcttc atctgtgcta tattcttcca
     1981 ttgtttcacc actaacaaag tggatgactc tccttgggac tttcttcttt tttcctatga
     2041 ctcccagttc tacattttca aagcctcttt cgttactcat ctgccctgca gattccccga
     2101 atgccgcggc ggccgcagct cccgaggctg cgacggcttc tcctcgttcc acaccgccca
     2161 ctggctcctg gtccatcgtc gtcgccacca cagggctgct ggcgctggtg agaaagaggg
     2221 taatggccgg taagcccact tccatggtgg cggatccgag ctcggtacca agcttaagtt
     2281 taaacgctag ccagcttggg tctccctata gtgagtcgta ttaatttcga taagccagta
     2341 agcagtgggt tctctagtta gccagagagc tctgcttata tagacctccc accgtacacg
     2401 cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg
     2461 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
     2521 tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat
     2581 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg
     2641 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
     2701 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     2761 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     2821 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     2881 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa
     2941 taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa aacgcctaac
     3001 cctaagcaga ttcttcatgc aattgtcggt caagccttgc cttgttgtag cttaaatttt
     3061 gctcgcgcac tactcagcga cctccaacac acaagcaggg agcagatact ggcttaacta
     3121 tgcggcatca gagcagattg tactgagagt gcaccatagg ggatcgggag atctcccgat
     3181 ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3241 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3301 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3361 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3421 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3481 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3541 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3601 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3661 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3721 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3781 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     3841 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     3901 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     3961 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     4021 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     4081 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4141 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4201 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4261 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4321 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4381 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4441 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4501 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4561 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4621 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4681 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4741 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4801 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4861 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4921 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4981 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5041 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5101 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5161 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5221 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5281 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5341 gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg ataagataca
     5401 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     5461 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttggggtgg
     5521 gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa
     5581 acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc
     5641 aggttgggcg tcgcttggtc ggtcatttcg aaccccagag tcccgctcag aagaactcgt
     5701 caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga
     5761 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta
     5821 tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc
     5881 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc
     5941 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct
     6001 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga
     6061 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc
     6121 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat
     6181 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga
     6241 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct
     6301 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg
     6361 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc
     6421 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca
     6481 tgcgaaacga tcctcatcct gtctcttgat cagatccgaa aatggatata caagctcccg
     6541 ggagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     6601 cagaggcaga ggcggcctcg gcctctgcat aaata
//
