LOCUS       sequence-266866-        8236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human 33-G-X5 in 293T cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8236)
  AUTHORS   Qin Y, Garrison BS, Ma W, Wang R, Jiang A, Li J, Mistry M, Bronson 
            RT, Santoro D, Franco C, Robinton DA, Stevens B, Rossi DJ, Lu C, 
            Springer TA
  TITLE     A Milieu Molecule for TGF-beta Required for Microglia Function in 
            the Nervous System.
  JOURNAL   Cell. 2018 Jun 28;174(1):156-171.e16. doi: 
            10.1016/j.cell.2018.05.027. Epub 2018 Jun 14.
  PUBMED    29909984
REFERENCE   2  (bases 1 to 8236)
  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: 
            "10.1016/j.cell.2018.05.027"; journalName: "Cell"; date: 
            "2018-06-28- 28"; volume: "174"; issue: "1"; pages: "156-171.e16"
COMMENT     Sequence Label: 33-G-X5-pEF1/V5-His
FEATURES             Location/Qualifiers
     source          1..8236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..32
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(151..171)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        156..485
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      336..471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     398..417
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             552..1346
                     /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(606..625)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1216..1235
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1522..1643
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1559..1578)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1613..1632
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1692..1708)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1692..1708)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1705..1727)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1716..1732
                     /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(1740..1746,1747..1764,1765..1770))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1785..1806
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1923..1940)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2083..2671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2163..2182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2842..3633,3634..3702))
                     /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     3465..3484
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3703..3807)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3882..3901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4189..5367
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4420..5358
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5315..5335
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     5384..5403
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5384..5402
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5522..5545
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7583..7624
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             7634..7651
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7674..7691)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7680..7904
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(7950..8236,1..142)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8037..8056)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
       61 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
      121 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
      181 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
      241 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
      301 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
      361 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
      421 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
      481 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
      541 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
      601 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
      661 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
      721 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
      781 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
      841 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
      901 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
      961 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1021 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     1081 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     1141 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     1201 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     1261 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     1321 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgcgaaat gaccgaccaa
     1381 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     1441 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     1501 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     1561 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     1621 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     1681 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     1741 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1801 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1861 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1921 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1981 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2041 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2101 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2161 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2221 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     2281 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     2341 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2401 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2461 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2521 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2581 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2641 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2701 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     2761 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2821 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2881 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2941 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3001 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3061 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3121 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3181 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3241 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     3301 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     3361 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     3421 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     3481 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     3541 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3601 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3661 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     3721 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     3781 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     3841 cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc
     3901 gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg
     3961 agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
     4021 agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga
     4081 ttattgacta ggcttttgca aaaagctttg caaagatgga taaagtttta aacagagagg
     4141 aatctttgca gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc
     4201 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     4261 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     4321 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     4381 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc
     4441 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
     4501 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
     4561 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
     4621 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     4681 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     4741 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     4801 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     4861 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     4921 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     4981 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     5041 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     5101 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     5161 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     5221 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     5281 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     5341 ttttttcttc catttcaggt gtcgtgagga attagcttgg tactaatacg actcactata
     5401 gggagaccca agctggctag gtaagcttgg taccgagctc ggatccgcta gcgccaccat
     5461 gtggtggcgc ctgtggtggc tgctgctgct gctgctgctg ctgtggccca tggtgtgggc
     5521 cgactacaag gacgacgacg acaagctcga ctggaggaac agaagcggaa cagccacagc
     5581 agcctcccaa ggagtctgca agttggtggg tggagccgct gactgccgag ggcagagcct
     5641 cgcttcggtg cccagcagcc tcccgcccca cgcccggatg ctcaccctgg atgccaaccc
     5701 tctcaagacc ctgtggaatc actccctcca gccttaccct ctcctggaga gcctcagcct
     5761 gcacagctgc cacctggagc gcatcagccg cggcgccttc caggagcaag gtcacctgcg
     5821 cagcctggta ctgggggaca actgcctctc agagaactac gaagagacgg cagccgccct
     5881 ccacgccctg ccgggcctgc ggaggctgga cttgtcagga aacgccctga cggaggacat
     5941 ggcagcgctc atgctccaga acctctcctc gctgcggtcc gtgtccctgg cggggaacac
     6001 catcatgcgg ctggacgact ccgtcttcga gggcctggag cgtctccggg agctggatct
     6061 gcagaggaac tacatcttcg agatcgaggg cggcgctttc gacggcctgg ctgagctgag
     6121 gcacctcaac ctggccttca acaacctccc ctgcatcgtg gacttcgggc tcacgcggct
     6181 gcgggtcctc aacgtcagct acaacgtcct ggagtggttc ctcgcgaccg ggggagaggc
     6241 tgccttcgag ctggagacgc tggacctgtc tcacaaccag ctgctgttct tcccgctgct
     6301 gccccagtac agcaagttgc ggaccctcct gctgcgcgac aacaacatgg gcttctaccg
     6361 ggacctgtac aacacctcgt cgccgaggga gatggtggcc cagttcctcc tcgtggacgg
     6421 caacgtgacc aacatcacca ccgtcagcct ctgggaagaa ttctcctcca gcgacctcgc
     6481 agatctccgc ttcctggaca tgagccagaa ccagttccag tacctgccag acggcttcct
     6541 gaggaaaatg ccttccctct cccacctgaa cctccaccag aattgcctga tgacgcttca
     6601 cattcgggag cacgagcccc ccggagcgct caccgagctg gacctgagcc acaaccagct
     6661 gtcggagctg cacctggctc cggggctggc cagctgcctg ggcagcctgc gcttgttcaa
     6721 cctgagctcc aaccagctcc tgggcgtccc ccctggcctc ttcgccaatg ctaggaacat
     6781 cactacactt gacatgagcc acaatcagat ctcactttgt cccctgccag ctgcctcgga
     6841 ccgggtgggc ccccctagct gtgtggattt caggaatatg gcatctttaa ggagcctgtc
     6901 tctggagggc tgtggcctgg gggcattgcc agactgccca ttccaaggga cctccctgac
     6961 ctacttagac ctctcaagca actggggggt tctgaatggg agcctcgccc cactccagga
     7021 tgttgccccc atgttacagg tcctgtctct caggaacatg ggcctccact ccagctttat
     7081 ggcgttggac ttctctgggt ttgggaatct cagggactta gatctgtcgg ggaattgctt
     7141 gaccaccttc ccaaggtttg ggggcagcct ggccctggag accctggatc tccgtagaaa
     7201 ctcgctcaca gcccttcccc agaaggctgt gtctgagcag ctctcgagag gtctgcggac
     7261 catctacctc agtcagaatc catatgactg ctgtggggtg gatggctggg gggccctgca
     7321 gcatgggcag acggtggccg actgggccat ggtcacctgc aacctctcct ccaagatcat
     7381 ccgcgtgacg gagctgcccg gaggtgtgcc tcgggactgc aagtggggcg gactgaagaa
     7441 catcaacctc atcatcatcc tcaccttcat actggtctct gccatcctcc tcaccacgct
     7501 ggccgcctgc tgctgcgtcc gccggcagaa gtttaaccaa cagtataaag cctaaagcgg
     7561 ccgctctaga gggcccttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc
     7621 tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac
     7681 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     7741 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     7801 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     7861 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     7921 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     7981 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     8041 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     8101 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     8161 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     8221 gacgttggag tccacg
//
