LOCUS       sequence-464147-        5981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Heterologous protein expression of glycine cleavage system H protein
            in Escherichia coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  TITLE     AI Proteins, Inc. plasmids
REFERENCE   2  (bases 1 to 5981)
  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: pAIP4_3A7L
FEATURES             Location/Qualifiers
     source          1..5981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(150..197)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     193..211
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(264..281)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(990..1019)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1042..1064
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1079..1103
                     /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     complement(1103..1122)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1104..1122)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1210..1229)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1435..1512
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1513..2595
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1532..1551)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3404..3595
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3589..3611)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3697..3836
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3752..3771
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3851..3868)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4022..4610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4102..4121)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4721..4740)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4732..5547
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4732..5547
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4805..4824)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5639..5981,1..113))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5776..5797)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
       61 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
      121 gccaatccgg atatagttcc tcctttcagc aaaaaacccc tcaagacccg tttagaggcc
      181 ccaaggggtt atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttcgg
      241 gctttgttag cagccggatc tcagtggtgg tggtggtggt gctcgagtca ttacgaaccc
      301 tcatcttcta agagggcttc gtacgcagtg gcatccagca gcgattccaa ttcgctctcg
      361 tcgctcgcct tgattttgaa aatccaaccg cctgcatacg gctcggagtt aaccaactcc
      421 gggctgtcgg acagcgcatc attcaccgct acaatttcac cgctaaccgg agcatagatg
      481 tcagatgccg ctttgacaga ttccgccaca gcgcagtcgt cgccagcgct gacggttgcg
      541 cccacctctg gcagatcaac aaaaaccatg tcacccagca gctcctgcgc atgttcggtg
      601 atgcccacgg tgtacgtacc atccgcctct ttacgcagcc attcgtgctc cttactatat
      661 tttagttcag cgggtacatt tgatccagag ccgccaatct gttcacggtg ggcttcaatg
      721 atgtcgttgt cttccatgtc caggtcttcc ggcgcttgat ctgcctggat gcgaataccg
      781 tcatacagga aacgcaggct atccatttcc ttgccctgac gtttggcaaa cgcttccatc
      841 aggcgacgca gcggcgtggt ctttttgatc ttaaagaaaa tttcagagct gccgtcacta
      901 actttcaggt taatatgggt ttccggtttc acttccggct tgacttccgg ctttgcttct
      961 tggttgactt ccgaatcttg caggctaccg tggtgatggt gatggtgatg atggtgatga
     1021 atcttcgcca tcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg
     1081 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     1141 cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     1201 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     1261 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     1321 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     1381 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     1441 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     1501 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     1561 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     1621 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     1681 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     1741 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     1801 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     1861 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     1921 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     1981 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     2041 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     2101 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     2161 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     2221 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     2281 attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc
     2341 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     2401 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     2461 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     2521 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     2581 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
     2641 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
     2701 catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt
     2761 gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat
     2821 cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg
     2881 tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccccacg
     2941 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     3001 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     3061 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     3121 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     3181 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     3241 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     3301 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     3361 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     3421 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     3481 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     3541 tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     3601 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     3661 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     3721 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3781 caccattgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     3841 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3901 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3961 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4021 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4081 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4141 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4201 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4261 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4321 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4381 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4441 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     4501 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4561 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4621 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4681 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
     4741 attcaacggg aaacgtcttg ctctaggccg cgattaaatt ccaacatgga tgctgattta
     4801 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
     4861 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     4921 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     4981 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg
     5041 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     5101 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     5161 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     5221 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     5281 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     5341 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     5401 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     5461 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     5521 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
     5581 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5641 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5701 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5761 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5821 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5881 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5941 cgatttagag cttgacgggg aaagccggcg aacgtggcga g
//
